Plant leaves originate from the shoot apical meristem (SAM) and undergo a developmental process of highly coordinated gene expression regulation. To date, only a few key regulators have been identified and characterised, so the gene expression cascades responsible for leaf cell specification and differentiation from SAM remain largely elusive. Here, we optimised a spatial transcriptomics protocol using the 10× Genomics Visium system and developed computational pipelines to reconstruct three-dimensional gene expression profiles of the SAM and sequentially developing leaves in maize seedlings. These enabled positional indexing of cells sampled from consecutive developmental stages, revealing dynamic transitions from undifferentiated stem cells in the SAM to functionally differentiated leaf structures. Through spatial-temporal transcriptome analysis, we identified distinct transcriptional programs and key regulatory genes involved in meristem maintenance, leaf primordia initiation, vascular tissue differentiation, and cellular heterogeneity. This approach outperforms the single-cell transcriptome profiling, which lacks temporal and spatial contexts. Our optimised experimental pipeline, which goes from section preparation to data processing, enables the spatial resolution and 3-dimensional mapping of gene expression profiles. The established pipeline is readily applicable to delineating molecular events underlying developmental transitions, cell type specifications, and differentiation in plants.

Understanding how isolated small populations persist and adapt in adverse environments is instrumental to evolutionary and conservation biology. We combine a chromosome-level genome assembly, population resequencing and forward-time simulations to reconstruct the history and viability of the Formosan landlocked salmon ( Oncorhynchus formosanus ), now restricted to a handful of high-mountain headwaters in Taiwan. We estimate that this lineage has diverged from Japanese masu salmon over one million years ago and has no detectable gene flow for ∼50,000 years. It has accumulated extensive chromosome fusions and expansions of cold-adaptation gene families, qualifying it as a new species rather than a subspecies of Japanese masu salmon. Whole-stream sampling reveals an overlooked Hehuan-Creek population that retains high heterozygosity and has gained unique alleles. Life-table simulations show that the Hehuan population has a notably lower extinction risk and can persist or even grow under low-to-moderate typhoon frequency, whereas Qijiawan-Creek population would decline precipitously under the same or higher frequency . These findings contradict the notion that peripheral populations are likely genetically depleted and support stream-specific “precision conservation” in place of broad, untargeted translocations that could erode local adaptation potential. Thus, our genomic-ecological analysis has uncovered hidden strong resilience in a critically endangered, climate-threatened salmonid lineage.

Introduction:Gibberellin 2-oxidases (GA2oxs) deactivate bioactive gibberellins and play essential roles in regulating rice growth and development. However, the relative contributions of individual OsGA2ox paralogs, their redundancy patterns, and dominant-minor relationships within this multigene family remain poorly understood. Clarifying these relationships is important for understanding how GA2ox genes coordinately influences plant development in rice. Methods:To address these questions, we generated 36 CRISPR/Cas9-edited rice lines targeting all nine OsGA2ox genes, including single-, double-, triple-, and quadruple-gene knockout mutants, and systematically analyzed their effects on agronomic traits to compare gene-specific and combinatorial functions. Results:In Class II, OsGA2ox1 is the main regulator of plant height, whereas OsGA2ox2 alone showed no discernable effect. Double mutants of these two genes showed a modest height increase and a reduced grain number but retained normal flowering time, indicating partial redundancy in regulating plant height and grain number, with OsGA2ox1 as the dominant contributor. In Class I, OsGA2ox3, OsGA2ox4, and OsGA2ox8 function as dominant suppressors of stem elongation, whereas OsGA2ox7 showed no phenotypical effect in single mutants but contributed to stem elongation control when disrupted together with other Class I members. The quadruple Class I mutant exhibited the greatest increase in plant height but reduced grain weight and fertility. In Class III, OsGA2ox5 and OsGA2ox6 regulated plant height, with OsGA2ox6 additionally influencing grain size, whereas OsGA2ox9 showed no detectable function. Discussion:These findings reveal a hierarchical but partially redundant regulatory framework within the rice GA2ox gene family, in which dominant and minor paralogs contribute unequally to GA-related growth regulation. Our study provides allele-validated genetic evidence for functional diversification among OsGA2ox paralogs and highlights how redundancy and specialization are integrated to stabilize hormone-regulated development and key agronomic traits in rice.

Biological nitrogen fixation (BNF), which is catalyzed by a large nitrogenase enzyme complex, has evolved in both bacteria and archaea. Indeed, nitrogen-fixing species are found in diverse living environments, and BNF has evolved even in aerobic bacteria, although the function of nitrogenase is inhibited by oxygen. BNF is, however, highly energy-costing, requiring 16 ATPs in a single nitrogen fixation reaction. To explain this paradox, we hypothesized that nitrogen-fixing species gain not only nitrogen-fixing ( nif ) genes but also non- nif genes to facilitate nitrogen fixation. We examined over 3500 nitrogen-fixing genomes and found that they have gained genes directly or indirectly related to BNF in diverse types of living environments, so that nitrogen-fixing species tend to have larger genomes than their non-nitrogen-fixing relatives. Interestingly, the non- nif genes gained tend to be located near nif -gene clusters, probably to achieve proximity effects such as coordinated gene regulation. For example, the most frequent among the genes gained are ABC transporter genes, which facilitate the absorption and physiological metabolism of carbon (e.g., sugars), nitrogen (e.g., amino acids), and trace elements (e.g., molybdenum), and many ABC transporter genes lie close to nif -gene clusters. From our findings, we propose the following scenario: BNF evolved in many archaea and bacteria because BNF is advantageous to its hosts, although it incurs a high energy cost. Then, gaining genes to facilitate BNF compensates the cost of BNF, facilitating the spread of nitrogen fixers to all living habitats. This expansion benefits the biosphere, as nitrogen is essential for all organisms.

Cellulosic biomass represents a promising feedstock for biofuel and biochemical production. However, its recalcitrant structure strongly hinders enzymatic degradation. Cellulosomes are large multienzyme complexes, highly efficient at degrading cellulose. A cellulase in a cellulosome has a dockerin domain that binds to a cohesin module on the CipA (cellulosome integrating protein A). In a native cellulosome all cohesins are identical, so that the cellulase types and their positions in a CipA cannot be controlled. Here, we constructed the largest designer CipA known to date. Using innovative techniques, we synthesized a designer CipA gene that encodes nine distinct cohesins and two cellulose-binding modules, which we named DCipA2B9C . Then, we fused nine distinct fungal cellulases separately with nine distinct dockerins for their precise positioning on DCipA2B9C to achieve enzyme proximity-effect. We constructed three yeast hosts to compare their performances. First, an enzyme host (EH) secretes nine dockerin-fused cellulases, including endoglucanases (EgIII-a, EgIII-m, and EgIII-c), exoglucanases (CBHII-j and EXG2-r), β-glucosidases (BGS-f and BGS-l), and cellulase boosters, including a LPMO-t and CDH-b. Second, the scaffoldin host (SH) expresses DCipA2B9C. Third, the cellulosome-9 host expresses DCipA2B9C and nine dockerin-fused cellulases. Native-PAGE and ELISA confirmed specific interactions between dockerins and cohesins. Additionally, native-PAGE, SDS-PAGE, and LC-MS verified the successful assembly of the multienzyme complex. Our performance evaluation showed that coculturing of EH and SH outperformed the cellulosome-9 host. It degraded microcrystalline cellulose efficiently to produce 14.29 g/L bioethanol, which surpassed all previously constructed yeast cellulosomes by fourfold or more. In summary, our study provides an effective approach to biomass degradation.

ABSTRACT The Nocardia genus represents a largely untapped source of valuable secondary metabolites, yet its biosynthetic potential, gene content, and evolutionary history remain underexplored. By analyzing 263 genomes across 88 species, we found that Nocardia varies greatly in genome size and gene content. It exhibits an open pangenome with a small core genome (<900 genes) and high genomic fluidity (0.76), indicating high gene turnover. A large proportion (75%) of its genes are species-specific, indicating high genomic plasticity. Average nucleotide identity (ANI) analysis confirmed taxonomic relationships, with most species showing ANI values (80–85%). N. globerula showed an ANI of ~84% with Rhodococcus erythropolis, supporting its reclassification under Rhodococcus. The biosynthetic capabilities of the Nocardia genus are striking, with the presence of >8,000 biosynthetic gene clusters (BGCs), dominated by type 1 polyketide synthase, terpenes, and non-ribosomal polypeptide synthetases, establishing Nocardia as the Actinomycetota genus that has the largest biosynthetic repertoire. Around 35% of BGCs remain uncharacterized, suggesting Nocardia’s high potential for novel natural product discoveries. Our study is the first to identify a prodigiosin BGC in Nocardia. Network analysis revealed complex evolutionary connections between Nocardia’s gene cluster families (GCFs) and MIBiG reference BGCs, suggesting evolutionary changes, including gene gains and losses, that may have influenced the genus’s BGC diversity and composition. Synteny analysis uncovered conserved and unique gene arrangements across Nocardia and related genera, mostly with core genes conserved in Actinomycetota. Our study addressed unmet clinical and biotechnological challenges while revealing evolutionary mechanisms that shape microbial diversity and adaptability.IMPORTANCEUnderstanding the genomic diversity and biosynthetic potential of microorganisms is instrumental for addressing issues in microbial evolution, natural product discovery, and host-microbe interactions. Nocardia, a bacterial genus known for its opportunistic pathogenicity, represents an underexplored group of immense genomic diversity and biosynthetic capabilities. This study employed genome mining to reveal the open pangenome of Nocardia and identified an extensive repertoire of BGCs, including novel clusters with the potential to produce therapeutically significant compounds such as prodigiosin-related compounds. By integrating genome mining, phylogenetics, and synteny analysis, this study provides insights into how genomic plasticity, species-specific genes, and evolutionary changes such as gene gains and losses that contribute to Nocardia's biosynthetic diversity and evolution. These findings contribute to advancing microbial genomics, evolution, and biotechnology by uncovering the potential of Nocardia to address challenges in infectious diseases and natural product discovery. This study exemplifies how genome mining can illuminate the ecological and clinical significance of microbial diversity.

Nitrogen is essential for all organisms, but biological nitrogen fixation (BNF) occurs only in a small fraction of prokaryotes. Previous studies divided nitrogenase-gene-carrying prokaryotes into Groups I to IV and provided evidence that BNF first evolved in bacteria. This study constructed a timetree of the evolution of nitrogen-fixation genes and estimated that archaea evolved BNF much later than bacteria and that nitrogen-fixing cyanobacteria evolved later than 1,900 MYA, considerably younger than the previous estimate of 2,200 MYA. Moreover, Groups III and II/I diverged ∼2,280 MYA, after the Kenorland supercontinent breakup (∼2,500-2,100 MYA) and the Great Oxidation Event (∼2,400-2,100 MYA); Groups III and Vnf/Anf diverged ∼2,086 MYA, after the Yarrabubba impact (∼2,229 MYA); and Groups II and I diverged ∼1,920 MYA, after the Vredefort impact (∼2,023 MYA). In summary, this study provided a timescale of BNF events and discussed the possible effects of geological events on BNF evolution.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We discovered that LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial -mesenchymal interactions for branching morphogenesis. ACTA2 compartments dermal papilla stem cells for feather cycling. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We found this primary feather transition largely conserved in chicken (precocious) and zebra finch (altricial) and discussed the possibility that this evolutionary adaptation process started in feathered dinosaurs.

One of the most intriguing traits found in domestic chickens is the Crest phenotype. This trait, characterized by a tuft of elongated feathers sprouted from the head, is found in breeds such as Polish chickens and Silkie chickens. Moreover, some crested chicken breeds also exhibit a protuberance in their anterodorsal skull region. Previous studies have strived to identify the causative factors of this trait. This study aimed to elucidate the role of chicken HOXC8 and HOXC10 in the formation of the Crest phenotype. We explored the effect of ectopic expression of HOXC8 or HOXC10 on the chicken craniofacial morphology using the RCAS retrovirus transformation system. Microcomputed tomography scanning was conducted to measure the 3D structure of the cranial bone of transgenic embryos for geometric morphometric analysis. We found that the ectopic expression of HOXC8 or HOXC10 in chicken heads caused mild morphological changes in the skull compared with the GFP-transgenic control group. Geometric morphometric analysis showed that HOXC8 and HOXC10 transgenic groups expressed a mild upward shape change in the frontal region of the skull compared with the control group, which is similar to what is seen in the crested chicken breeds. In conclusion, this study supports findings in previous studies in which HOX genes play a role in the formation of the altered skull morphology related to the Crest phenotype. It also supports that mutations in HOX genes may contribute to intra- and inter-specific variation in morphological traits in vertebrates.

To understand the mechanism underlying the evolution of SARS-CoV-2 in a population, we sequenced 92 viral genomes from Assam, India. Analysis of these and database sequences revealed a complete selective sweep of a haplotype in Assam carrying 13 pre-existing variants, including a high leap in frequency of a variant on ORF8, which is involved in immune evasion. A comparative study between sequences of same lineage and similar time frames in and outside Assam showed that 10 of the 13 pre-existing variants had a frequency ranging from 96 to 99%, and the remaining 3 had a low frequency outside Assam. Using a phylogenetic approach to infer sequential occurrences of variants we found that the variant Phe120del on ORF8, which had a low frequency (1.75%) outside Assam, is at the base of the phylogenetic tree of variants and became totally fixed (100%) in Assam population. Based on this observation, we inferred that the variant on ORF8 had a selective advantage, so it carried the haplotype to reach the100% frequency. The haplotype also carried 32 pre-existing variants at a frequency from 1.00 to 80.00% outside Assam. Those of these variants that are more closely linked to the S-protein locus, which often carries advantageous mutations and is tightly linked to the ORF8 locus, retained higher frequencies, while the less tightly linked variants showed lower frequencies, likely due to recombination among co- circulating variants in Assam. The ratios of non-synonymous substitutions to synonymous substitutions suggested that some genes such as those coding for the S-protein and non-structural proteins underwent positive selection while others were subject to purifying selection during their evolution in Assam. Furthermore, we observed negative correlation of the Ct value of qRT-PCR of the patients with abundant ORF6 transcripts, suggesting that ORF6 can be used as a marker for estimating viral titer. In conclusion, our in-depth analysis of SARS-CoV-2 genomes in a regional population reveals the mechanism and dynamics of viral evolution.

Lactobacillus plantarum is known for its probiotics benefit to host, although the effects vary among strains. This study conducted a feeding experiment of three Lactobacillus strains, MRS8, MRS18 and MRS20, which were isolated from kefir and incorporated into the diets of shrimp to evaluate the effects of non-specific immunity, immune-related gene expression, and disease resistance of white shrimp (Penaeus vannamei) against Vibrio alginolyticus. To prepare the experimental feed groups, the basic feed was mixed with different concentrations of L. plantarum strains MRS8, MRS18, and MRS 20, which were incorporated at 0 CFU (control), 1 × 106 CFU (groups 8-6, 18-6, and 20-6), and 1 × 109 CFU (groups 8-9, 18-9, and 20-9) per gram of diet for an in vivo assay. During the rearing period for 28 days of feeding each group, immune responses, namely the total hemocyte count (THC), phagocytic rate (PR), phenoloxidase activity, and respiratory burst were examined on days 0, 1, 4, 7, 14, and 28. The results showed that groups 20-6, 18-9 and 20-9 improved THC, and groups 18-9 and 20-9 improved phenoloxidase activity and respiratory burst as well. The expression of immunity-related genes was also examined. Group 8-9 increased the expression of LGBP, penaeidin 2 (PEN2) and CP, group 18-9 increased the expression of proPO1, ALF, Lysozyme, penaeidin 3 (PEN3) and SOD, and group 20-9 increased the expression of LGBP, ALF, crustin, PEN2, PEN3, penaeidin 4 (PEN4) and CP (p < 0.05). Groups 18-6, 18-9, 2-6, and 20-9 were further used in the challenge test. After feeding for 7 days and 14 days, Vibrio alginolyticus was injected into white shrimp and observed the shrimp survival for 168 h. The results showed that compared to the control, all groups improved the survival rate. Especially, feeding group 18-9 for 14 days improved the survival rate of white shrimp (p < 0.05). After the challenge test for 14 days, the midgut DNA of survival white shrimps was extracted to analyze the colonization of L. plantarum. Among the groups, (6.61 ± 3.58) × 105 CFU/pre shrimp of L. plantarum in feeding group 18-9 and (5.86 ± 2.27) × 105 CFU/pre shrimp in group 20-9 were evaluated by qPCR. Taken together, group 18-9 had the best effects on the non-specific immunity, the immune-related gene expression, and the disease resistance, which might be due to the benefit of the probiotic colonization.

The rice GA biosynthetic gene OsGA3ox1 has been proposed to regulate pollen development through the gametophytic manner, but cellular characterization of its mutant pollen is lacking. In this study, three heterozygotic biallelic variants, "-3/-19", "-3/-2" and "-3/-10", each containing one null and one 3bp-deletion allele, were obtained by the CRISPR/Cas9 technique for the functional study of OsGA3ox1. The three homozygotes, "-19/-19", "-2/-2" and "-10/-10", derived from heterozygotic variants, did not affect the development of most vegetative and floral organs but showed a significant reduction in seed-setting rate and in pollen viability. Anatomic characterizations of these mutated osga3ox1 pollens revealed defects in starch granule accumulation and pollen wall development. Additional molecular characterization suggests that abnormal pollen development in the osga3ox1 mutants might be linked to the regulation of transcription factors OsGAMYB, OsTDR and OsbHLH142 during late pollen development. In brief, the rice GA3ox1 is a crucial gene that modulates pollen starch granule accumulation and pollen wall development at the gametophytic phase.

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Ｗe develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels.

Horizontal gene transfer (HGT) is a means of exchanging genetic material asexually. The process by which horizontally transferred genes are domesticated by the host genome is of great interest but is not well understood. In this study, we determined the telomere-to-telomere genome sequence of the wheat-infecting Pyricularia oryzae strain Br48. SNP analysis indicated that the Br48 strain is a hybrid of wheat- and Brachiaria-infecting strains by a sexual or parasexual cross. Comparative genomic analysis identified several megabase-scale "insertions" in the Br48 genome, some of which were possibly gained by HGT-related events from related species, such as P. pennisetigena or P. grisea. Notably, the mega-insertions often contained genes whose phylogeny is not congruent with the species phylogeny. Moreover, some of the genes have a close homolog even in distantly related organisms, such as basidiomycetes or prokaryotes, implying the involvement of multiple HGT events. Interestingly, the levels of the silent epigenetic marks H3K9me3 and H3K27me3 in a genomic region tended to be negatively correlated with the phylogenetic concordance of genes in the same region, suggesting that horizontally transferred DNA is preferentially targeted for epigenetic silencing. Indeed, the putative HGT-derived genes were activated when MoKmt6, the gene responsible for H3K27me3 modification, was deleted. Notably, these genes also tended to be up-regulated during infection, suggesting that they are now under host control and have contributed to establishing a fungal niche. In conclusion, this study suggests that epigenetic modifications have played an important role in the domestication of HGT-derived genes in the P. oryzae genome.

The superior photosynthetic efficiency of C4leaves over C3leaves is owing to their unique Kranz anatomy, in which the vein is surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. Kranz anatomy development starts from three contiguous ground meristem (GM) cells, but its regulators and underlying molecular mechanism are largely unknown. To identify the regulators, we obtained the transcriptomes of 11 maize embryonic leaf cell types from five stages of pre-Kranz cells starting from median GM cells and six stages of pre-M cells starting from undifferentiated cells. Principal component and clustering analyses of transcriptomic data revealed rapid pre-Kranz cell differentiation in the first two stages but slow differentiation in the last three stages, suggesting early Kranz cell fate determination. In contrast, pre-M cells exhibit a more prolonged transcriptional differentiation process. Differential gene expression and coexpression analyses identified gene coexpression modules, one of which included 3 auxin transporter and 18 transcription factor (TF) genes, including known regulators of Kranz anatomy and/or vascular development. In situ hybridization of 11 TF genes validated their expression in early Kranz development. We determined the binding motifs of 15 TFs, predicted TF target gene relationships among the 18 TF and 3 auxin transporter genes, and validated 67 predictions by electrophoresis mobility shift assay. From these data, we constructed a gene regulatory network for Kranz development. Our study sheds light on the regulation of early maize leaf development and provides candidate leaf development regulators for future study.

The mandarin duck, Aix galericulata, is popular in East Asian cultures and displays exaggerated sexual dimorphism, especially in feather traits during breeding seasons. We generated and annotated the first mandarin duck de novo assembly, which was 1.08 Gb in size and encoded 16,615 proteins. Using a phylogenomic approach calibrated with fossils and molecular divergences, we inferred that the last common ancestor of ducks occurred 13.3-26.7 Ma. The majority of the mandarin duck genome repetitive sequences belonged to the chicken repeat 1 (CR1) retroposon CR1-J2_Pass, which underwent a duck lineage-specific burst. Synteny analyses among ducks revealed infrequent chromosomal rearrangements in which breaks were enriched in LINE retrotransposons and DNA transposons. The calculation of the dN/dS ratio revealed that the majority of duck genes were under strong purifying selection. The expanded gene families in the mandarin duck are primarily involved in olfactory perception as well as the development and morphogenesis of feather and branching structures. This new reference genome will improve our understanding of the morphological and physiological characteristics of ducks and provide a valuable resource for functional genomics studies to investigate the feather traits of the mandarin duck.

Chloroplasts are the sites for photosynthesis, and two Golden2-like factors act as transcriptional activators of chloroplast development in rice (Oryza sativa L.) and maize (Zea mays L.). Rice OsGLK1 and OsGLK2 are orthologous to maize ZmGLK1 (ZmG1) and ZmGLK2 (ZmG2), respectively. However, while rice OsGLK1 and OsGLK2 act redundantly to regulate chloroplast development in mesophyll cells, maize ZmG1 and ZmG2 are functionally specialized and expressed in different cell-specific manners. To boost rice chloroplast development and photosynthesis, we generated transgenic rice plants overexpressing ZmG1 and ZmG2, individually or simultaneously, with constitutive promoters (pZmUbi::ZmG1 and p35S::ZmG2) or maize promoters (pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2). Both ZmG1 and ZmG2 genes were highly expressed in transgenic rice leaves. Moreover, ZmG1 and ZmG2 showed coordinated expression in pZmG1::ZmG1/pZmG2::ZmG2 plants. All Golden2-like (GLK) transgenic plants had higher chlorophyll and protein contents, Rubisco activities and photosynthetic rates per unit leaf area in flag leaves. However, the highest grain yields occurred when maize promoters were used; pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2 transgenic plants showed increases in grain yield by 51%, 47%, and 70%, respectively. In contrast, the pZmUbi::ZmG1 plant produced smaller seeds without yield increases. Transcriptome analysis indicated that maize GLKs act as master regulators promoting the expression of both photosynthesis-related and stress-responsive regulatory genes in both rice shoot and root. Thus, by promoting these important functions under the control of their own promoters, maize GLK1 and GLK2 genes together dramatically improved rice photosynthetic performance and productivity. A similar approach can potentially improve the productivity of many other crops.

The origin of nitrogen fixation is an important issue in evolutionary biology. While nitrogen is required by all living organisms, only a small fraction of bacteria and archaea can fix nitrogen. The prevailing view is that nitrogen fixation first evolved in archaea and was later transferred to bacteria. However, nitrogen-fixing (Nif) bacteria are far larger in number and far more diverse in ecological niches than Nif archaea. We, therefore, propose the bacteria-first hypothesis, which postulates that nitrogen fixation first evolved in bacteria and was later transferred to archaea. As >30,000 prokaryotic genomes have been sequenced, we conduct an in-depth comparison of the two hypotheses. We first identify the six genes involved in nitrogen fixation in all sequenced prokaryotic genomes and then reconstruct phylogenetic trees using the six Nif proteins individually or in combination. In each of these trees, the earliest lineages are bacterial Nif protein sequences and in the oldest clade (group) the archaeal sequences are all nested inside bacterial sequences, suggesting that the Nif proteins first evolved in bacteria. The bacteria-first hypothesis is further supported by the observation that the majority of Nif archaea carry the major bacterial Mo (molybdenum) transporter (ModABC) rather than the archaeal Mo transporter (WtpABC). Moreover, in our phylogeny of all available ModA and WtpA protein sequences, the earliest lineages are bacterial sequences while archaeal sequences are nested inside bacterial sequences. Furthermore, the bacteria-first hypothesis is supported by available isotopic data. In conclusion, our study strongly supports the bacteria-first hypothesis.

Previous reviews have already explored the safety and bioavailability of astaxanthin, as well as its beneficial effects on human body. The great commercial potential in a variety of industries, such as the pharmaceutical and health supplement industries, has led to a skyrocketing demand for natural astaxanthin. In this study, we have successfully optimized the astaxanthin yield up to 12.8 mg/g DCW in a probiotic yeast and purity to 97%. We also verified that it is the desired free-form 3S, 3'S configurational stereoisomer by NMR and FITR that can significantly increase the bioavailability of astaxanthin. In addition, we have proven that our extracted astaxanthin crystals have higher antioxidant capabilities compared with natural esterified astaxanthin from H. pluvialis. We also screened for potential adverse effects of the pure astaxanthin crystals extracted from the engineered probiotic yeast by dosing SD rats with 6, 12, and 24 mg/kg/day of astaxanthin crystals via oral gavages for a 13-week period and have found no significant biological differences between the control and treatment groups in rats of both genders, further confirming the safety of astaxanthin crystals. This study demonstrates that developing metabolically engineered microorganisms provides a safe and feasible approach for the bio-based production of many beneficial compounds, including astaxanthin.

Sustainable management of harvested hyperaccumulating plants used in phytoremediation of toxic metals is considered quite challenging. The biomass of hyperaccumulators can be an attractive feedstock for bioethanol production, while removal of toxic metals and efficient ethanol conversion systems are necessary to ensure safe and optimum bioethanol production. This study aimed to develop a compatible and efficient consolidated bioprocessing system (CBP) to produce bioethanol from phytomass of arsenic (As)-hyperaccumulator Pteris vittata. A recombinant kefir yeast Kluyveromyces marxianus (KR7 strain) contained six cellulase genes and two recombinant Bacillus subtilis (Type 1 and Type 2 strains) carrying cellulosomal genes in different arrangements were used, and the saccharification and fermentation of P. vittata phytomass were investigated through one-step (KR7 strain only) and co-culture (B. subtilis + KR7 strain) methods. The results of reducing sugar showed that KR7 strain possesses higher saccharification ability than both B. subtilis strains. While the co-culture of B. subtilis Type 2 strain with KR7 strain gave the highest ethanol yield, which achieved 48.5% ethanol yield from P. vittata phytomass after 3 d cultivation. In addition, simple pretreatment by shaking P. vittata with 1% of HNO3 efficiently removed more than 80% of As from the phytomass, suggesting its applicability and feasibility for efficient As extraction to ensure efficient and non-toxic ethanol production. Our results confirm that the developed recombinant bacteria-yeast co-culture possesses a great potential for optimum bioethanol production from As-hyperaccumulators-derived phytomass via CBP.