Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra

11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011beta-hydroxylase (P45011beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads, P45011beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.     

Growth of engineered Pseudomonas putida KT2440 on glucose,xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

Lignocellulosic biomass is the most abundant bioresource on earth containing polymers mainly consisting of d ‐glucose, d ‐xylose, l ‐arabinose, and further sugars. In order to establish this alternative feedstock apart from applications in food, we engineered Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of xylose and arabinose in addition to glucose as sole carbon sources. The d ‐xylose‐metabolizing strain P. putida KT2440_xylAB and l ‐arabinose‐metabolizing strain P. putida KT2440_araBAD were constructed by introducing respective operons from Escherichia coli. Surprisingly, we found out that both recombinant strains were able to grow on xylose as well as arabinose with high cell densities and growth rates comparable to glucose. In addition, the growth characteristics on various mixtures of glucose, xylose, and arabinose were investigated, which demonstrated the efficient co‐utilization of hexose and pentose sugars. Finally, the possibility of using lignocellulose hydrolysate as substrate for the two recombinant strains was verified. The recombinant P. putida KT2440 strains presented here as flexible microbial biocatalysts to convert lignocellulosic sugars will undoubtedly contribute to the economic feasibility of the production of valuable compounds derived from renewable feedstock.     

Recent Advances in Genome-wide Analyses of Plant Potassium Transporter Families

Plants require potassium (K⁺) as a macronutrient to support numerous physiological processes. Understanding how this nutrient is transported, stored, and utilized within plants is crucial for breeding crops with high K⁺ use efficiency. As K⁺ is not metabolized, cross-membrane transport becomes a rate-limiting step for efficient distribution and utilization in plants. Several K⁺ transporter families, such as KUP/HAK/KT and KEA transporters and Shaker-like and TPK channels, play dominant roles in plant K⁺ transport processes. In this review, we provide a comprehensive contemporary overview of our knowledge about these K⁺ transporter families in angiosperms, with a major focus on the genome-wide identification of K⁺ transporter families, subcellular localization, spatial expression, function and regulation. We also expanded the genome-wide search for the K⁺ transporter genes and examined their tissue-specific expression in Camelina sativa, a polyploid oil-seed crop with a potential to adapt to marginal lands for biofuel purposes and contribution to sustainable agriculture. In addition, we present new insights and emphasis on the study of K⁺ transporters in polyploids in an effort to generate crops with high K⁺ Utilization Efficiency (KUE).     

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO₂-eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.     

Biotransformation of corn bran derived ferulic acid to vanillic acid using engineered Pseudomonas putida KT2440

Abstract

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the vanAB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95?±?1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87?±?0.38% molar yield. With fermentation time of less than 24?h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.     

Genome-Wide Identification and Expression Profiling of the Shaker K⁺ Channel and HAK/KUP/KT Transporter Gene Families in Grape (<i>Vitis vinifera</i> L.)

Potassium (K⁺) is an essential macronutrient for plants to maintain normal growth and development. Shaker-like K⁺ channels and HAK/KUP/KT transporters are critical components in the K⁺ acquisition and translocation. In this study, we identified 9 Shaker-like K⁺ channel (VvK) and 18 HAK/KUP/KT transporter (VvKUP) genes in grape, which were renamed according to their distributions in the genome and relative linear orders among the distinct chromosomes. Similar structure organizations were found within each group according to the exon/intron structure and protein motif analysis. Chromosomal distribution analysis showed that 9 VvK genes and 18 VvKUP genes were unevenly distributed on 7 or 10 putative grape chromosomes. Three pairs of tandem duplicated genes and one pair of segmental duplicated genes were observed in the expansion of the grape VvKUP genes. Gene expression omnibus (GEO) data analysis showed that VvK and VvKUP genes were expressed differentially in distinct tissues. Various cis-acting regulatory elements pertinent to phytohormone responses and abiotic stresses, including K⁺ deficiency response and drought stress, were detected in the promoter region of VvK and VvKUP genes. This study provides valuable information for further functional studies of VvK and VvKUP genes, and lays a foundation to explore K⁺ uptake and utilization in fruit trees.     

41种植物精油对淡色库蚊的熏蒸活性

为发现具有较高杀蚊活性的精油, 推动植物精油类卫生杀虫剂的创制, 本研究采用三角瓶熏蒸法测定了17科41种植物精油对淡色库蚊Culex pipiens pallens雌成蚊的熏蒸活性, 并进一步采用密闭圆筒法验证其杀虫毒力大小。三角瓶熏蒸法测定表明, 在12 μL/L浓度下, 留兰香油（spearmint oil）、 薰衣草油（lavender oil）、 山苍子油（Litsea cubeba oil）等26种精油均有较强的熏蒸作用, KT₅₀小于15 min。该26种精油用密闭圆筒熏蒸法进一步测定表明, 在浓度为10 μL/L时, 冬青油（wintergreen oil）、 艾叶油（blumea oil）、 薄荷油（peppermint oil）、 桉叶油（Eucalyptus robusta oil）和蓝桉油（E. globulus oil）等植物精油熏蒸活性较强, 其KT₅₀分别为16.91, 21.20, 22.57, 18.43和19.48 min。结果证明冬青油、 艾叶油、 薄荷油、 桉叶油和蓝桉油等5种精油对淡色库蚊具有较强的熏蒸活性, 具备作为淡色库蚊防控剂开发的潜力, 值得进一步研究。     

KT和Ca2+对绿豆下胚轴原生质体的体积和CaM含量的影响

以黄化绿豆幼苗下胚轴原生质体为材料,探讨钙信使系统在KT诱导原生质体体积变化中的作用。lμmol/L KT可诱导含钙培养液中绿豆下胚轴原生质体膨大,处理后30min达到最大体积。Ca2 通道阻断剂Verapamil、Ca2 通道竞争性抑制剂LaCl3和钙调素拮抗剂TFP、CPZ可明显抑制KT诱导的原生质体膨大。另一方面,无论是KT处理还是对照(CaCl2单独处理),原生质体内CaM含量均在处理后30min时达到峰值,前者是后者的5倍．在KT CaCl2处理液中加入5μmol/L．Verapamil、50μmol/L LaCl3、5μmol/L,TFP或CPZ后原生质体内CaM含量都大大降低。以上结果表明,CaM可能与Ca2 共同参与KT的信号传递。     

pH-stat fed-batch process to enhance the production of cis, cis-muconate from benzoate by Pseudomonas putida KT2440-JD1

Pseudomonas putida KT2440-JD1 is able to cometabolize benzoate to cis, cis-muconate in the presence of glucose as growth substrate. P. putida KT2440-JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis-muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis-muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis-muconate. To prevent overdoses of benzoate, a pH-stat fed-batch process for the production of cis, cis-muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH-stat fed-batch process was 0.6 g (4.3 mmol) g dry cell weight(-1) h(-1), whereas 18.5 g L(-1) cis, cis-muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH-stat fed-batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motility.