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The economic feasibility and waste treatment problem are challenges to the industrialization of lycopene production from Saccharomyces cerevisiae. In this study, fermentation wastewater, biomass residue, and residual D-galactose are recycled for lycopene production. Results show that when fresh water is totally replaced by wastewater, lycopene titer attains 1.21 ± 0.02 g/L, which is 14.2% higher than the fresh water group (P < 0.05). An 80% replacement ratio of yeast extract by biomass residue causes no significant difference to lycopene production while 100% replacement ratio significantly lowers lycopene titer compared with the yeast extract group. Then, a novel fermentation medium containing wastewater and biomass residue with supplementing 3 g/L yeast extract and D-galactose is used for lycopene production. Lycopene titer increases 22.4% than the traditional fermentation in shake flasks (P < 0.05). Continuous self-cycling strategy using wastewater and biomass residue was tested in shake flasks. The mean lycopene titer of the first five recycles shows no significant difference with the start batch. Scaling up to 70 L fermenter, the mean lycopene titer attains 5.88 ± 0.15 g/L in three recycles, which is 22.25% higher than the start batch (P < 0.05). Economic analysis shows that the lowest unite product cost is achieved when four recycles are accomplished, which is 29.6% lower than the traditional fermentation while the chemical oxygen demand decreases 64.0%. Our study shows that continuous self-cycling fermentation process for lycopene production is feasible for the first time. The comprehensive utilization of wastewater and biomass residue from lycopene production by S. cerevisiae and achievement of high lycopene titer will hopefully accelerate industrialization of microbial production of lycopene.

The reproductive process is usually controlled by the hypothalamic-pituitary-gonad axis in vertebrates, while Kiss/gonadotropin-releasing hormone (GnRH) system in the hypothalamus is required for mammalian reproduction but dispensable for fish reproduction. The regulation of follicle stimulating hormone/luteinizing hormone (LH) expression in fish species is still unknown. Here, we identified miR-200s on chromosome 23 (chr23-miR-200s) as important regulators for female zebrafish reproduction. Knockout of chr23-miR-200s (chr23-miR-200s-KO) resulted in dysregulated expression of luteinizing hormone beta lhb (luteinizing hormone beta) and some hormone genes in the pituitary as revealed by comparative transcriptome profiling, leading to failure of oocyte maturation and ovulation as well as defects in reproductive duct development. Chr23-miR-200s mainly expressed in the pituitary and regulated lhb expression by targeting the transcription repressor wt1a. Injection of human chorionic gonadotropin (hCG) could rescue the defects of oocyte maturation in chr23-miR-200s-KO zebrafish, whereas GnRH or LHRH-A2 could not, suggesting that Chr23-miR-200s regulated lhb expression in a GnRH-independent pathway. It was remarkable that either injection of carp pituitary extraction, or co-injection of hCG with synthetic oxytocin and vasotocin could greatly rescue the defects of both oocyte maturation and ovulation in chr23-miR-200s-KO zebrafish. Altogether, our findings highlight an important function of chr23-miR-200s in controlling oocyte maturation by regulation LH expression, and oxytocin and vasotocin are potentially responsible for the ovulation in fish species.

The sex-determining genes are found to be variable among different fish species. Yellow catfish (Pelteobagrus fulvidraco) is an important aquaculture fish species in China with XX/XY sex-determining type. Recently, YY super-male yellow catfish has been successfully produced by combining hormonal-induced sex reversal method with sex chromosome-linked markers. Here, we identified a novel PDZ domain-containing gene in yellow catfish designated as pfpdz1, in whose intron the sex-linked marker was located. The coding sequence of pfpdz1 in Y chromosome was identical to that in X chromosome except a missense SNP (A/T) that changes an amino acid (E8V) in the N-terminal region. Pfpdz1 displayed male-specific expression during sex differentiation. Overexpression of pfpdz1 using additive transgenesis induces XX ovary to differentiate into testis-like tissue, while the targeted inactivation of pfpdz1 in Y chromosome using CRISPR/Cas9-mediated mutagenesis triggers ovarian differentiation. Furthermore, we demonstrated that pfpdz1 initiates testicular differentiation through upregulating expression of amh, dmrt1 and sox9a1, as well as downregulating expression of cyp19a1, foxl2 and wnt4. Our data provide functional evidence that pfpdz1 is significant for male differentiation and maintenance in yellow catfish.

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of growth hormone (GH) signaling in somatic growth of mammalian. However, the corresponding homologue gene of Stat5b is unknown in fish species. In this study, we generated loss-of-function mutants in stat5.1 and stat5.2, two stat5 homologues existing in zebrafish. In stat5.1-deficient zebrafish, a significant reduction of body length and body weight was detected in the embryos/larvae and adults compared with the wild-type control fish, and sexual size dimorphism in adult zebrafish was also eliminated. However, the stat5.2-deficient zebrafish displayed a normal developmental phenotype during all lifespan. Chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) method was adopted to further investigate the potential transcriptional targets of Stat5 protein and cast much light upon the biological function of Stat5. We identified more than 800 genes as transcriptional targets of Stat5 during zebrafish embryogenesis. KEGG analysis indicated that the Stat5 target gene network is predominantly linked to the metabolic pathways, neuroactive ligand-receptor interaction and JAK-STAT signaling pathways. Further validation studies suggested that Stat5.1 protein could directly regulate the expression of gh1, and stat5.1-mutated zebrafish showed a reduction of gh1 mRNA level. In the present study, stat5.1 was revealed as the corresponding homologue gene of Stat5b in fish species. Additionally, we found a novel molecular interaction between Stat5.1/Stat5b and GH, and unraveled a positive feedback loop Stat5.1-GH-Stat5.1 which is necessary for somatic growth and body development in zebrafish.

STAT (Signal Transducers and Activators of Transcription) gene family members have been revealed to be involved in cell growth and differentiation in vertebrates. Despite their physiological importance, their functions are poorly studied at organ and systemic levels. In this study, we performed a genome-wide analysis using data from invertebrates to vertebrates to identify STAT genes and analyze their evolutionary history. Interestingly, the STAT gene family undergoes genome duplications during the evolutionary history with STAT3 homologues firstly appearing in the basal extant vertebrate, sea lamprey, suggesting its possible roles in spine formation. To investigate the functions of stat3 in fish species, TALEN technology was performed to generate mutant zebrafish lines. Stat3 mutant zebrafish showed no obvious defects at early developmental stage but displayed severe lateral and vertical curvature of the spine (scoliosis), spine fracture and the incomplete bone joints with narrower junction between vertebrae at early juvenile stage, as indicated by Alizarin red and Alcian blue staining, radiography and micro-computed tomography (MicroCT) analysis. Transcriptome analysis reveals dramatic alterations in a number of genes involved in immune and infection response, skeletal development and somatic growth, especially downregulated expression of collagen gene family, in the juvenile stat3 mutant zebrafish. Moreover, most of the collagen genes were detected to have abnormal expression pattern during the formation of spine deformities in stat3 mutants. Our data reveal that stat3 is specially expressed in vertebrates and required for normal spine development and immune function in zebrafish.

Sexual size dimorphism is an interesting phenomenon occurred in many fish species. Wildtype zebrafish exhibits a significant sexual dimorphism in body size at the adult stage. Previous studies indicated that sexual size dimorphism was eliminated in stat5b-mutated zebrafish. Herein, the comparative transcriptome analysis was conducted to observe the genes and pathways involved in sexual size dimorphism. The number of male-biased and female-biased genes was much less in the liver of stat5b mutant zebrafish than in wildtype. Gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that multiple pathways related to metabolism were affected upon loss of stat5b function. qRT-PCR results also validated that sexually dimorphic expression of a set of genes was lost when stat5b was mutated. Furthermore, the weighted correlation network analysis (WGCNA) detected many candidate genes related to the growth traits and stat5b function, such as greb1, lepr, and igf2b. Our data suggest that stat5b should regulate the sexually dimorphic gene expression in zebrafish liver and add in understanding of the molecular mechanisms underlying sexual size dimorphism in fish species.

Besides its well-documented roles in cell proliferation, apoptosis, and carcinogenesis, the function of the p53-microRNA axis has been recently revealed in the reproductive system. Recent studies indicated that miR-200 family members are dysregulated in nonobstructive azoospermia patients, whereas their functions remain poorly documented. The aim of this study was to investigate the function of the miR-200 family on zebrafish testis development and sperm activity. There was no substantial difference in testis morphology and histology between wild-type (WT) and knockout zebrafish with deletion of miR-200 cluster on chromosome 6 (chr6-miR-200-KO) or on chromosome 23 (chr23-miR-200-KO). Interestingly, compared with WT zebrafish, the chr6-miR-200-KO zebrafish had no difference on sperm motility, whereas chr23-miR-200-KO zebrafish showed significantly improved sperm motility. Consistently, ectopic expression of miR-429a, miR-200a, and miR-200b, which are located in the miR-200 cluster on chromosome 23, significantly reduced motility traits of sperm. Several sperm motility-related genes, such as amh, wt1a, and srd5a2b have been confirmed as direct targets of miR-200s on chr23. 17α-ethynylestradiol (EE2) exposure resulted in upregulated expression of p53 and miR-429a in testis and impairment of sperm motility. Strikingly, in p53 mutant zebrafish testis, the expression levels of miR-200s on chr23 were significantly reduced and accompanied by a stimulation of sperm motility. Moreover, the upregulation of miR-429a associated with EE2 treatment was abolished in testis with p53 mutation. And the impairment of sperm activity by EE2 treatment was also eliminated when p53 was mutated. Together, our results reveal that miR-200 cluster on chromosome 23 controls sperm motility in a p53-dependent manner.

Increasing attention has been focused on the role of microRNAs in post-transcription regulation during spermatogenesis. Recently, the miR-34 family has been shown to be involved in the spermatogenesis, but the clear function of the miR-34 family in spermatogenesis is still obscure. Here we analyzed the function of miR-34a, a member of the miR-34 family, during spermatogenesis using miR-34a knockout zebrafish generated by the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system. miR-34a knockout zebrafish showed no obvious defects on testis morphology and sperm quantity. However, we found a significant increase in progressive sperm motility that is one of the pivotal factors influencing in vitro fertilization rates, in the knockout zebrafish. Moreover, breeding experiments showed that, when miR-34a-knockout male zebrafish mated with the wide-type females, they had a higher fertilization rate than did the wide-type males. Glycogen synthase kinase-3a (gsk3a), a potential sperm motility regulatory gene was predicted to be targeted by miR-34a, which was further supported by luciferase reporter assays, since a significant decrease of luciferase activity was detected upon ectopic overexpression of miR-34a. Our findings suggest that miR-34a downregulates gsk3a by targeting its 3′ untranslated region, and miR-34a/gsk3a interaction modulates sperm motility in zebrafish. This study will help in understanding in the role of the miR-34 family during spermatogenesis and will set paths for further studies.

Sexual size dimorphism is the consequence of differential expression of sex-biased genes related to feeding and growth. Leptin is known to regulate energy balance by regulating food intake. In order to investigate the molecular mechanism of sexual size dimorphism in yellow catfish (Pelteobagrus fulvidraco), the expression of leptin (lep) and its functional receptor (lepr) were detected during larval development. Both lep and lepr have lower expression in males than in females during 1–4 weeks post hatching. 17a-Methyltestosterone (MT) treatment resulted in decreased expression of lep and lepr in both male and female larval fish. Interestingly, the mRNA levels of lep and lepr in juvenile male were significantly decreased compared with juvenile female during short-term fasting periods. Lep was predicted to be a potential target of miR-200a and miR-200b that had an opposite expression pattern to lep in male and female larvas. The results of luciferase reporter assay suggested that lep is a target of miR-200a/-200b. Subsequently, male hormone and fasting treatment have opposite effects on the expression of miR-200a/-200b and lep between males and females. In summary, our results suggest that sexual size dimorphism in fish species is probably caused by the sexually dimorphic expression of leptin, which could be negatively regulated by miR-200a/-200b.

Background 21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity. Results In this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known. Conclusions Heterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0720-y) contains supplementary material, which is available to authorized users.

Dmrt2b (doublesex and mab-3 related transcription factor 2b) has been revealed to be involved in zebrafish slow muscle development. However, the function of dmrt2a, a paralogue gene of dmrt2b, remains unclear during zebrafish muscle development. Here, we demonstrated that knockdown of dmrt2a resulted in severe developmental defects, and caused downregulation of fast muscle marker myhz-2 and upregulation of slow muscle marker myhz-5, respectively. It is known that microRNAs (miRNAs) control many biological events including muscle development. Dmrt2a was predicted to be a target gene of miR-203, which was further verified by luciferase reporter assay, since miR-203a was found to directly reduce the expression of dmrt2a by binding to the seed sequence of its 3'UTR. After miR-203a injection into zebrafish embryos, the expression of dmrt2a was significantly inhibited. Similar to the effect of dmrt2a knockdown, miR-203a overexpression led to downregulation of myhz-2 and upregulation of myhz-5. Our studies indicated that miR-203a directly regulated dmrt2a expression to control fast and slow muscle differentiation, while overexpression of miR-203a or knockdown of dmrt2a will impair fast muscle development and promote slow muscle development.