Recent advances in keratinase production via protein engineering,breeding,and fermentation

The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes.Microbial keratinases can convert keratin wastes into valuable compounds for mankind.Since the market for keratinases cannot be satisfied by production from wild hosts,it is obligatory to develop hosts with high keratinase yields.The intention of this review is to evaluate microbial keratinase advancement through protein engineering,breeding techniques,and fermentation optimization.The main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis methods.Moreover,modifying the expression elements and host engineering are also two unique ways to augment the keratinase yield.Intending to accelerate the production of modified keratinase,this review attempts to highlight the optimization of expression elements,such as promoter engineering,UTR,signal peptide,and codon optimization.Moreover,the approaches of host engineering including strengthening precursor supply,membrane surface engineering,and optimization of secretion pathways were also explained here.Furthermore,it is also essential to optimize the medium composition and fermentation condition for high keratinase yield.This review also addressed the present advancements,difficulties,and tendencies in the field of microbial keratinase production,along with its potential.

Porcine Breeding Management in a Large-scale Piggery with Microbial Fermentation Bed

[Objective] The behavior of eating, drinking, defecating and peeing of 1 500 pigs in a large-scale microbial fermentation bed-equipped piggery was observed. We hoped to find some simple indicators that could reflect the health status of swinery and to provide experience for the swinery performance management in large-scale microbial fermentation bed-equipped piggery. [Method] The body weight （BW）, daily BW gain, feed intake and other indicators of different-day-old pigs were recorded in details. Based on the recorded data, the models between BW, BW gain, average daily feed intake and feed/gain ratio and growth days （d） were established. In addition, the incidences of pox-like macula （dermatitis）, diarrhea （gastrointestinal disease）, cough （respiratory disease）, stiff pig （malnutrition）, conjunctivitis （eye disease） and foot inflection （trauma） among fattening pigs were also investigated. [Result] The BW range, average BW, daily BW gain, breeding days, daily feed intake range, average daily feed intake, staged feed intake, accumulated feed intake, feed/gain ratio and accumulated feed/gain ratio of different-day-old pigs were studied, respectively. Four dynamic models were established for the growth of pigs： （1） the BW （y）-age （x） mod- el： y=0.758 9x-19.883 （3=0.993 7）; （2） the BW gain （y）-age （x） model： y=1.039 5x05051 （F=0.885 4）; （3） the average daily feed intake （y）-age （x） model： y=0.023 5x-0.334 3 （F=0.991 7）; （4） the feed/gain ratio （y）-age （x） model： y=0.022x＋0.427 8 （P=0.988 5）. Based on these models, the corresponding theoretical growth value of pigs at different growth stage could be predicted. The main diseases occurred among the swinery in the large-scale microbial fermentation bed piggery included pox-like macula （dermatitis）, diarrhea （gastrointestinal disease）, cough （respiratory disease）, stiff pig （mal- nutrition）, conjunctivitis （eye disease） and foot inflection （trauma）. The deadly infec- tious diseases had been not found among the pigs. [Conclusion] When the actual BW, BW gain, average daily feed intake and feed/gain ratio were all lower than the theoretical values predicted by the models, the management should be enhanced. The average daily feed intake of 60 to 65-day-old pigs was lower than the theoretic value, indicating that the pigs could not adapt nicely to the fermentation bed at the very early stage. When the pigs grew up to 70 to 75 d old, the average daily feed intake was higher than the theoretical value, indicating that the pigs had adapted to the fermentation bed. In particularly, average daily feed intake of 75-day-old pigs was higher than the theoretical value by 21%. It was suggested the fermentation bed was conducive to the growth of pigs. Considering the occurrence of diseases among pigs, the overall incidence was relatively low. The incidence of each disease was all lower than 10% with little difficulty in treating. If the management of mattress was strength- ened, such as paying attention to feeding and keeping water clean, many diseases could heal by themselves.

Piglets Behavior Characteristics under Breeding Mode of Microbial Fermentation Bed

[Objective] This experiment aimed to compare piglets behavior under microbial fermentation bed breeding mode and traditional rearing mode. [Method] Pig behavior＇s statistical analysis under both microbial fermentation bed pig breeding mode and traditional rearing mode was se lected to study the effect of different breeding environment on pig＇s growth [ Result] The results showed that the duration of piglets lying and sleeping behavior accounted for the highest proportion of tested behavior, up to more than 70% ,followed by feeding and drinking which reached 15.96% （under microbial fermentation bed breeding mode） and 9.33% （under traditional rearing mode） respectively. Compared with traditional intensive rearing mode, piglet exploratory behavior＇s occurrence probability and duration increased significantly, by 28.62% and 12.21% respectively. Fighting behavior＇s occurrence probability and duration decreased significantly, by 49.83% and 91.26% respectively. Nutrient index and health index were introduced to evaluate piglet＇s general heath condition under microbial fermentation bed breeding mode, and results indicated that the health index of piglets under microbial fermentation bed breeding mode had slight difference from that under traditional mode, namely 10.83 and 10.03 respectively. But health index of piglets under microbial fermentation bed breeding mode was 245.12, much higher than 21.96 under traditional mode. [ Conclusion] Piglets would be healthier under microbial fermentation bed breeding mode.

Breeding of Coenzyme Q_(10) Produced Strain by Low-Energy Ion Implantation and Optimization of Coenzyme Q_(10) Fermentation

In order to increase the production efficiency of coenzyme Q10, the original strain Agrobacterium tumefaciens ATCC 4452 was mutated by means of Nitrogen ions implantation. A mutant strain, ATX 12, with high contents of coenzyme Q10 was selected. Subsequently, the conditions such as carbohydrate concentration, nitrogen source concentration, inoculum＇s size, seed age, aeration and temperature which might affect the production of CoQ10 were investigated in detail. Under optimal conditions, the maximum concentration of the intracellular CoQ10 reached 200.3 mg/L after 80 h fed-batch fermentation, about 245% increasing in CoQ10 production after ion implantation, compared to the original strain.

Drought-Tolerant Rice at Molecular Breeding Eras:An Emerging Reality

Rice(Oryza sativa L.)stands as the most significantly influential food crop in the developing world,with its total production and yield stability affected by environmental stress.Drought stress impacts about 45%of the world’s rice area,affecting plants at molecular,biochemical,physiological,and phenotypic levels.The conventional breeding method,predominantly employing single pedigree selection,has been widely utilized in breeding numerous drought-tolerant rice varieties since the Green Revolution.With rapid progress in plant molecular biology,hundreds of drought-tolerant QTLs/genes have been identified and tested in rice crops under both indoor and field conditions.Several genes have been introgressed into elite germplasm to develop commercially accepted drought-tolerant varieties,resulting in the development of several drought-tolerant rice varieties through marker-assisted selection and genetically engineered approaches.This review provides up-to-date information on proof-of-concept genes and breeding methods in the molecular breeding era,offering guidance for rice breeders to develop drought-tolerant rice varieties.

Breeding and Seed Reproduction Techniques of a New Tetraploid Common Buckwheat Variety‘Pintianqiao 3’

[Objectives]This study was conducted to actively carry out the breeding of new tetraploid common buckwheat varieties and its supporting breeding techniques.[Methods]Pintianqiao 3 is a new tetraploid common buckwheat variety developed by College of Agriculture of Shanxi Agricultural University and Agricultural Genetic Resources Center of Shanxi Agricultural University,using‘Pintianqiao 1’as the parent,through mutation treatment with 0.2%colchicine aqueous solution,grain selection,plant selection,isolation and identification,variety comparison,regional test and field investigation.The variety has chromosomes 2n=4X=32,and shows a spring sowing period of 101 d and a summer sowing period of 80 d,large flowers and seeds(with a 1000-grain weight of 41.4 g),and good resistance to lodging.[Results]From 2021 to 2022,Pintianqiao 3 participated in the independent joint regional test of common buckwheat varieties in Shanxi Province,and the average yield in 10 test positions was 1.8 kg,equivalent to 1800 kg/hm^(2),which was 8.4%higher than the control.It passed the field investigation conducted by Shanxi provincial expert group for identification of non-major crop varieties in Dongyang and Kelan experimental sites on September 2-3,2022.On January 4,2024,it passed the preliminary examination of Shanxi Provincial Crop Variety Approval Committee.The seed reproduction technique of Pintianqiao 3 including land selection,preparation before sowing,sowing,field management and timely harvesting has been developed.[Conclusions]This study provides technical support for the demonstration and popularization of this new variety.

Long-term trends in the abundance and breeding performance in Adélie penguins:the Argentine Ecosystem Monitoring Program

In this work,we report long-term trends in the abundance and breeding performance of Adélie penguins(Pygoscelis adeliae)nesting in three Antarctic colonies(i.e.,at Martin Point,South Orkneys Islands;Stranger Point/Cabo Funes,South Shetland Islands;and Esperanza/Hope Bay in the Antarctic Peninsula)from 1995/96 to 2022/23.Using yearly count data of breeding groups selected,we observed a decline in the number of breeding pairs and chicks in crèche at all colonies studied.However,the magnitude of change was higher at Stranger Point than that in the remaining colonies.Moreover,the index of breeding success,which was calculated as the ratio of chicks in crèche to breeding pairs,exhibited no apparent trend throughout the study period.However,it displayed greater variability at Martin Point compared to the other two colonies under investigation.Although the number of chicks in crèche of Adélie penguins showed a declining pattern,the average breeding performance was similar to that reported in gentoo penguin colonies,specifically,those undergoing a population increase(even in sympatric colonies facing similar local conditions).Consequently,it is plausible to assume a reduction of the over-winter survival as a likely cause of the declining trend observed,at least in the Stranger Point and Esperanza colonies.However,we cannot rule out local effects during the breeding season affecting the Adélie population of Martin Point.

Corrigendum to“Migration pattern of a population of Barn Swallows(Hirundo rustica)breeding in East Asian tropical region”[Avian Res.15(2024)100192]

The authors regret that the caption of Fig.4 should be replaced as below.

Process Analysis of Mobile Fermentation of Spent White Hypsizygus marmoreus Substrate with Self-made Mobile Fermentation Bag

The self-made mobile fermentation bag for spent white Hypsizygus marmoreus substrate was developed, and the relevant fermentation process was studied. The results showed that under the condition of single addition of Lactobacillus,nitrogen-free extract was degraded into lactic acid, leading to decreased pH value.On day 10, the fermentation effect reached the best with Lactobacillus abundance of 5.12×10~7/ml, lactic acid content of 0.48% and strong acid flavor. At this time, the fermentation material was moist without mildew and agglomeration, and was suitable for livestock and poultry. However, after 10 days, undesirable acids and mildew generated, a large amount of lactic acid bacteria died, and the fermentation material turned black and agglomerated, and became unsuitable for feeding livestock and poultry. Throughout the fermentation process, the pH value first decreased continuously until to 4.0, and then remained stable. During the fermentation of spent white H. marmoreus substrate, the nitrogen-free extract and crude fiber contents decreased, the crude protein content increased, while other indicators remained unchanged.

Creating large EMS populations for functional genomics and breeding in wheat

Wheat germplasm is a fundamental resource for basic research,applied studies,and wheat breeding,which can be enriched normally by several paths,such as collecting natural lines,accumulating breeding lines,and introducing mutagenesis materials.Ethyl methane sulfonate(EMS)is an alkylating agent that can effectively introduce genetic variations in a wide variety of plant species.In this study,we created a million-scale EMS population(MEP)that started with the Chinese wheat cultivars‘Luyan 128’,‘Jimai 38’,‘Jimai 44’,and‘Shannong 30’.In the M1 generation,the MEP had numerous phenotypical variations,such as>3,000 chlorophyll-deficient mutants,2,519 compact spikes,and 1,692 male sterile spikes.There were also rare mutations,including 30 independent tillers each with double heads.Some M1 variations of chlorophyll-deficiency and compact spikes were inheritable,appearing in the M2 or M3 generations.To advance the entire MEP to higher generations,we adopted a single-seed descendent(SSD)approach.All other seed composites of M2 were used to screen other agronomically important traits,such as the tolerance to herbicide quizalofop-P-methyl.The MEP is available for collaborative projects,and provides a valuable toolbox for wheat genetics and breeding for sustainable agriculture.

Structure and immunomodulatory activity of Lentinus edodes polysaccharides modified by probiotic fermentation

Plant-based fermentations provide an untapped source for novel biotechnological applications.In this study,a probiotic named Lactobacillus fermentum 21828 was introduced to ferment Lentinus edodes.Polysaccharides were extracted from fermented and non-fermented L.edodes and purified via DEAE-52 and Sephadex G-100.The components designated F-LEP-2a and NF-LEP-2a were analyzed by FT-IR,HPGPC,HPAEC,SEM,GC-MS and NMR.The results revealed that probiotic fermentation increased the molecular weight from 1.16×10^(4) Da to 1.87×10^(4) Da and altered the proportions of glucose,galactose and mannose,in which glucose increased from 45.94%to 48.16%.Methylation analysis and NMR spectra indicated that F-LEP-2a and NF-LEP-2a had similar linkage patterns.Furthermore,their immunomodulatory activities were evaluated with immunosuppressive mice.NF-LEP and F-LEP improved immune organ indices,immunoglobulin(Ig G and Ig M)and cytokines concentrations;restored the antioxidation capacity of liver;and maintained the balance of gut microbiota.F-LEP displayed better moderating effects on the spleen index,immunoglobulin,cytokines and the diversity of gut microbiota than NF-LEP(200,400 mg/kg).Our study provides an efficient and environment-friendly way for the structural modification of polysaccharides,which helps to enhance their biological activity and promote their wide application in food,medicine and other fields.

Rice Heat Tolerance Breeding: A Comprehensive Review and Forward Gaze

The yield potential of rice is seriously affected by heat stress due to climate change. Since rice is a staple food globally, it is imperative to develop heat-resistant rice varieties. Thus, a thorough understanding of the complex molecular mechanisms underlying heat tolerance and the impact of high temperatures on various critical stages of the crop is needed. Adoption of both conventional and innovative breeding strategies offers a long-term advantage over other methods, such as agronomic practices, to counter heat stress. In this review, we summarize the effects of heat stress, regulatory pathways for heat tolerance, phenotyping strategies, and various breeding methods available for developing heat-tolerant rice. We offer perspectives and knowledge to guide future research endeavors aimed at enhancing the ability of rice to withstand heat stress and ultimately benefit humanity.

The Advantages of Methane Production by Combined Fermentation of Lignite and Wheat Straw

Biogasification of coal is important for clean utilization of coal. Experiments on the fermentation of single lignite, single straw and their mixture were performed to explore the variation characteristics of gas production potential, microbial community and methanogenic metabolic pathways of mixture. Research has shown that mixed fermentation of lignite and straw significantly promoted biomethane production. The abundance of hydrolytic acidifying functional bacteria genera (Sphaerochaeta, Lentimicrobium) in mixed fermentation was higher than that in the fermentation of single lignite and single straw. The abundance of some key CAZy metabolic enzyme gene sequences in mixed fermentation group was increased, which was favorable to improve methane production. Aceticlastic methanogenesis was the most critical methanogenic pathway and acetic acid pathway was more competitive in methanogenic mode during peak fermentation. Macrogenomics provided theoretical support for the claim that mixed fermentation of coal and straw promoted biomethane metabolism, which was potentially valuable in expanding methanogenesis from mixed fermentation of lignite with different biomasses.

Karachi provides a good opportunity for low-cost generation advancement of cotton for breeding and seed purity

Cotton is said to be the backbone of Pakistan's economy.Cotton production is facing many challenges such as climate change,pests and diseases,and competition from food crops(Ali et al.,2019).One of the major issues faced by cotton production is seed purity,as cotton is often cross-pollinated,therefore breeders are hard to maintain seed purity.For example,non-Bt cotton varieties are often contaminated with Bt seeds which is an important limiting factor.Another important consideration in cotton breeding is rapid generation advancement.

Postbiotics from Saccharomyces cerevisiae fermentation stabilize microbiota in rumen liquid digesta during grain-based subacute ruminal acidosis(SARA) in lactating dairy cows

Background Subacute ruminal acidosis(SARA)is a common metabolic disorder of high yielding dairy cows,and it is associated with dysbiosis of the rumen and gut microbiome and host inflammation.This study evaluated the impact of two postbiotics from Saccharomyces cerevisiae fermentation products(SCFP)on rumen liquid associated microbiota of lactating dairy cows subjected to repeated grain-based SARA challenges.A total of 32 rumen cannulated cows were randomly assigned to 4 treatments from 4 weeks before until 12 weeks after parturition.Treatment groups included a Control diet or diets supplemented with postbiotics(SCFPa,14 g/d Original XPC;SCFPb-1X,19 g/d Nutri Tek;SCFPb-2X,38 g/d Nutri Tek,Diamond V,Cedar Rapids,IA,USA).Grain-based SARA challenges were conducted during week 5(SARA1)and week 8(SARA2)after parturition by replacing 20%DM of the base total mixed ration(TMR)with pellets containing 50%ground barley and 50%ground wheat.Total DNA from rumen liquid samples was subjected to V3–V416S r RNA gene amplicon sequencing.Characteristics of rumen microbiota were compared among treatments and SARA stages.Results Both SARA challenges reduced the diversity and richness of rumen liquid microbiota,altered the overall composition(β-diversity),and its predicted functionality including carbohydrates and amino acids metabolic pathways.The SARA challenges also reduced the number of significant associations among different taxa,number of hub taxa and their composition in the microbial co-occurrence networks.Supplementation with SCFP postbiotics,in particular SCFPb-2X,enhanced the robustness of the rumen microbiota.The SCFP supplemented cows had less fluctuation in relative abundances of community members when exposed to SARA challenges.The SCFP supplementation promoted the populations of lactate utilizing and fibrolytic bacteria,including members of Ruminococcaceae and Lachnospiraceae,and also increased the numbers of hub taxa during non-SARA and SARA stages.Supplementation with SCFPb-2X prevented the fluctuations in the abundances of hub taxa that were positively correlated with the acetate concentration,andα-andβ-diversity metrics in rumen liquid digesta.Conclusions Induction of SARA challenges reduced microbiota richness and diversity and caused fluctuations in major bacterial phyla in rumen liquid microbiota in lactating dairy cows.Supplementation of SCFP postbiotics could attenuate adverse effects of SARA on rumen liquid microbiota.

Microbial Diversity and Key Metabolic Pathways in Lignite-Promoted Anaerobic Fermentation with Residual Sludge

To enhance methane production efficiency in lignite anaerobic digestion and explore new ways for residual sludge utilization, this study employed the co-fermentation of lignite and residual sludge for biomethane conversion. The bacterial colony structure, metabolic pathways, and interactions between residual sludge and lignite in anaerobic methanogenic fermentation with different mass ratios were analyzed using macrogenomics sequencing. This study aimed to explore the mechanisms involved in the co-anaerobic fermentation of lignite and residual sludge. The results indicated that the addition of sludge enhanced the metabolic pathways in hydrolysis acidification, hydrogen-acetic acid production, and methanation phases. Notably, the enhancement of acetate- and carbon dioxide-nutrient metabolic pathways was more pronounced, with increased activity observed in related enzymes such as acetic acid kinase (k00925) and acetyl coenzyme synthetase (K01895). This increased enzymatic activity facilitated the microbial conversion of biomethane. The results of the study indicated that the sludge exhibited a promotional effect on the methane produced through the anaerobic fermentation of lignite, providing valuable insights for lignite and residual sludge resource utilization.

Topgrafting as a tool in operational Scots pine breeding

Effective breeding requires multiplying desired genotypes,keeping them at a convenient location to perform crosses more efficiently,and building orchards to generate material for reforestation.While some of these aims can be achieved by conventional grafting involving only rootstock and scion,topgrafting is known to deliver all in a shorter time span.In this study,Scots pine scions were grafted onto the upper and lower tree crowns in two clonal archives with the aim of inducing early female and male strobili produc-tion,respectively.Their survival rates and strobili production were analyzed with generalized linear mixed models.Sur-vival was low(14%)to moderate(41%),and mainly affected by the topgraft genotype,interstock genotype,crown posi-tion and weather conditions in connection with the grafting procedure.Survival was not affected by the cardinal position in the crown(south or north).Male flowering was ample three years after grafting and reached 56%in the first year among live scions,increasing to 62 and 59%in consecutive years.Female flowering was scarce and was 9%at first,later increasing to 26 and 20%of living scions but was strongly affected by the topgraft genotype.In one subset of scions,female flowering was observed 1 year after grafting.Overall,flowering success was mainly affected by the topgraft and interstock genotypes,and secondary growth of scions.This is one of few reports on topgrafting in functional Scots pine clonal archives.

Influence of nitrogen status on fermentation performances of non-Saccharomyces yeasts:a review

Nitrogen,one of the most crucial nutrients present in grapes and musts,plays a key role in yeast activities during alcoholic fermentation.Such influences are imposed on yeast growth and fermentation performances including the formation of secondary metabolites.Saccharomyces cerevisiae,the main yeast responsible for fermentation,has been studied extensively regarding nitrogen impacts.On the other hand,a similar study for non-Saccharomyces yeasts,whose contributions to winemaking have gradually been acknowledged,remains to be fully explored,with a few studies being reported.This review starts by discussing nitrogen impacts on non-Saccharomyces yeast growth and fermentation kinetics in different case scenarios,then proceeds to summarize the nitrogen preferences of individual yeast strains with regulation mechanisms elucidated by recent studies.Detailed discussions on the influences on the production of volatile compounds and proposed pathways therein are made,followed by future work suggested as the final section.In summarizing the nitrogen impacts on non-Saccharomyces yeasts throughout alcoholic fermentation,this review will be helpful in obtaining a more comprehensive view on these non-conventional wine yeasts in terms of nutrient requirements and corresponding volatile production.Research gaps will therefore be elucidated for future research.

Efficient Breeding of Early-Maturing Rice Cultivar by Editing Hd6 via CRISPR/Cas9

Rice heading date,or flowering time,is important for yield optimization and regional adaptation.While many genes controlling this trait in rice are known,breeders often need only slight adjustments.Our study used the CRISPR/Cas9 method to edit the Hd6 gene in the early-maturing,late-season japonica rice cultivar Nanjing 46(NJ46),creating three mutants with reduced flowering times of 9-12 d under natural conditions in Nanjing city,Jiangsu Province,China.These mutants showed higher Hd3a and RFT1 expression levels without compromising yield or eating and cooking quality,demonstrating that Hd6 gene editing is an effective precision breeding tool for shortening heading date without laborious traditional methods.

The Influence of Gratering, Fermentation and Variety on the Physio-Chemical Quality of Cassava Starch

The study was conducted to determine the influence of gratering and fermentation parameters on the physicochemical quality of starch obtained from two cassava varieties in Sierra Leone (i.e., SLICASS 11 and SLICASS 6). Fresh cassava roots harvested from the Department of Agricultural Engineering were peeled, washed and grated before fermentation and starch extraction. Fermentation was carried out under separate aerobic and anaerobic conditions for a period of Seven days. Physicochemical analyses were conducted at the Postharvest Food and Bioprocess Engineering Laboratory of the Department of Agricultural and Bio-System Engineering, Njala University to determine the solubility, water absorption capacity and swelling power of starch extracts obtained from various experimental treatments. Fermentation method and duration had significant effects on the solubility, water absorption capacity (WAC) and swelling power (SP) of starch extracts. Maximum solubility and WAC were recorded on the fifth day, for both cassava varieties tested, with apparent significant difference resulting from the two gratering bands (i.e., with 1.5-inch nail hole and 4-inch nail hole sizes, respectively). While swelling power increased consistently with temperature for starch obtained from SLICASS-11 variety, an irregular pattern was observed for SLICASS-6 variety. A multiple correlation analysis proposes a significant and weak correlation between temperature, WAC (+0.150) and swelling power (+0.048). Also multiple correlation analyses suggest a significant correlation between fermentation period, the functional properties of starch extracts obtained from both fermentation methods and cassava varieties (i.e., solubility (−0.226), water absorption capacity (+0.301) and swelling power (+0.329)).