Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop fo...

Understanding the molecular basis for controlled H 2 O 2 activation is of fundamental importance for peroxide-driven catalysis by metalloenzymes. In addition to O 2 activation in the presence of stoichiometric reductants, an increasing number of metalloenzymes are found to activate the H 2 O 2 cosubstrate for oxidative transformations in the absence of stoichiometric reductants. Herein, we characterized the X-ray structure of the P450BM3 F87A mutant in complex with the dual-functional small molecule (DFSM) N-(ω-imidazolyl)-hexanoyl-Lphenylalanine (Im-C6-Phe), which enables an efficient peroxygenase activity for P450BM3. Our computational investigations show that the H 2 O 2 activations by P450BM3 are highly dependent on the substrate and the DFSM. In the absence of both the substrate and the DFSM, H 2 O 2 activation via the O−O homolysis mechanism is significantly inhibited by the H-bonding network from the proximal H of H 2 O 2 . However, the presence of the substrate expels the solvation waters and disrupts the H-bonding network from the proximal H of H 2 O 2 , thus remarkably favoring homolytic O−O cleavage toward Cpd I formation. However, the presence of the DFSM forms a proton channel between the imidazolyl group of the DFSM and the proximal H of H 2 O 2 , thus enabling a heterolytic O−O cleavage and Cpd I formation that is greatly favored over the homolysis mechanism. Meanwhile, our simulations demonstrate that the H-bonding network from the distal H of H 2 O 2 is the key to control of the H 2 O 2 activation in the homolytic route. These findings are in line with all available experimental data and highlight the key roles of H-bonding networks in dictating H 2 O 2 activations.

An alginate lyase-producing bacterial strain, Pseudoalteromonas sp. SM0524, was screened from marine rotten kelp. In an optimized condition, the production of alginate lyase from Pseudoalteromonas sp. SM0524 reached 62.6 U/mL, suggesting that strain SM0524 is a good producer of alginate lyases. The bifunctional alginate lyase aly-SJ02 secreted by strain SM0524 was purified. Aly-SJ02 had an apparent molecular mass of 32 kDa. The optimal temperature and pH of aly-SJ02 toward sodium alginate was 50 °C and 8.5, respectively. The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C. Aly-SJ02 was most stable at pH 8.0. N-terminal sequence analysis suggested that aly-SJ02 may be an alginate lyase of polysaccharide lyase family 18. Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-d-mannuronic acid), indicating that it is a bifunctional alginate lyase. Aly-SJ02 had lower Km toward polyG than toward polyM and sodium alginate. Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.

Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics.

N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for was evaluated in by targeting and, two essential genes for acetate and butyrate production, respectively. and single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P , we achieved a mutation rate of 75% for deletion without serial subculturing as suggested previously for Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. An efficient CRISPR-Cas9 genome engineering system was developed for N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.

Background:The maturation and catalysis mechanisms of the PL18 alginate lyases have not yet been reported. Results:The N-terminal extension in the precursor of PL18, aly-SJ02, helped the catalytic domain fold correctly. Key residues for substrate recognition and catalysis were determined. Conclusion:The catalytic mechanism of aly-SJ02 is proposed. Significance: This study provides the foremost insight into maturation and catalysis of PL18 alginate lyases.

Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum. The system consisting of CreHI (for installation of a phosphate directing/ anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p-and m-alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.alkylphenol | selective oxidation | chemomimetic biocatalysis | cytochrome P450 enzymes | Corynebacterium glutamicum A lkylphenols, p-, m-, and o-alkylated phenols, e.g., 1-10, (Fig. 1A) are among the most important synthetic precursors for manufacturing a vast variety of chemical products including detergents, polymers, lubricants, antioxidants, emulsifiers, pesticides, and pharmaceuticals (1). Alkylphenols are also priority environmental pollutants because they are toxic, xenoestrogenic, or carcinogenic to wildlife and humans (2, 3). The selective oxidation of the aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates for synthesizing more downstream products (4), but also in biological degradation of these environmentally hazardous compounds (5). In particular, some oxidized alkylphenols are direct structural motifs in drugs (e.g., metoprolol) (6) and bioactive ingredients of medicinal plants (Fig. 1B).Chemically, these oxidative transformations have been practiced for a long time by using superoxides, organocatalyst, high valence transition metals, or strong oxidants such as nitric acid, chromic acid, and potassium permanganate (4,7,8). However, a number of problems are persistently associated with the chemical oxidation of aliphatic C-H bonds in alkylphenols: (i) the requirement of protection/deprotection of the phenolic hydroxyl group under reaction conditions that are distinct from those for oxidation complicates the synthetic process; (ii) it is difficult to delicately control the extent of oxidations (i.e., alcohol vs. aldehyde/ketone vs. carboxylic acid); (iii) the oxidation of an aromatic C-H bond may sometimes occur when using strong oxidants; (iv) the regio-and stereoselective oxidation of an unactivated sp 3 C-H bond (in alkylphenols) remains a central challenge despite recent advances in combined use of specialized directing groups with transition met...

Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65), Psychromonas (10/65) and Polaribacter (8/65) were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases.