Transcriptome analysis of ovarian maturation in a chondrostei Chinese sturgeon Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) with an evolutionary history of over 200 million years, has a long lifespan, and an extremely late and asynchronous sexual maturation (8-18 years for males and 14-26 years for females), resulting in the difficulty of mature adult culture. However, little is known about the regulatory mechanisms of the transition among ovarian maturation stages in the Chinese sturgeon. We performed de novo transcriptome sequencing of the Chinese sturgeon at different ovarian maturation stages (FII, FIII, and FIV). The number of differentially expressed genes (DEGs) between FII and FIII/FIV (33,517/34,217) was more than that between FIII and FIV (22,123), suggesting that the transition from FII to FIII/FIV is more important than that from FIII to FIV for ovarian maturation. The number of upregulated genes was more than that of the downregulated genes, suggesting that increased gene expression was involved in the transition from FII to FIII/FIV. The representative pathways of DEGs were steroid biosynthesis, fatty acid biosynthesis, fatty acid elongation, glycerolipid metabolism, biosynthesis of unsaturated fatty acid, and α-linolenic acid metabolism. The differential expressions from the transcriptome sequencing were validated with real-time reverse-transcription polymerase chain reaction. We also found 13 genes in sexual development, female sex determination, gonadal development, ovarian maturation, ovarian follicle development, and oocyte development pathways, which were differently expressed among fish at FII, FIII, and FIV. We suggest that enhanced synthesis of steroid, unsaturated fatty acid, and α-linolenic acid may contribute to ovarian maturation of the Chinese sturgeon. These potential determinants provide a glimpse of the molecular architecture of ovary development in sturgeons.

Identification and sexually dimorphic expression of vasa isoforms in Dabry's sturgeon (Acipenser dabryanus), and functional analysis of vasa 3'-untranslated region.

Germ cells are set aside from somatic cells early in embryogenesis, and are responsible for transmitting genetic information through generations. Vasa is a highly conserved germ cell marker across animal phyla, and widely used to label primordial germ cells. Dabry's sturgeon is a rare and endangered species distributed solely in the Yangtze River basin. Here, seven vasa isoforms, named Advasa1-7, were isolated and characterized in Dabry's sturgeon. RT-PCR and western blot analyses revealed that vasa mRNA and protein were mainly restricted to the testis and ovary, but exhibited sexually dimorphic expression. Cellular and subcellular localization uncovered that Advasa mRNA and protein displayed mitotic and meiotic expression in females, and mainly showed mitotic expression in males; surprisingly, they exhibited both cytoplasmic and nuclear expression in the ovarian germ cells, while showing exclusively cytoplasmic expression in the testicular germ cells. By microinjecting chimeric RNA consisting of the red fluorescent protein coding region and the Advasa 3'-untranslated region into embryos of Dabry's sturgeon, zebrafish and medaka, we demonstrated that it had the ability to visualize primordial germ cells (PGCs) in Dabry's sturgeon and zebrafish but not in medaka. It seemed that the machinery of vasa 3'UTR RNA localization was conserved between Dabry's sturgeon and ostariophysan, while possibly changed during the divergence of euteleosts and ostariophysan. Finally, Dabry's sturgeon PGCs moved on the yolk ball, and migrated toward the genital ridge via mesenchyme. Taken together, these results provide new information for vasa expression pattern and function, and lay a foundation for PGC cryopreservation and conservation of Dabry's sturgeon.

Differential expression of fertility genes boule and dazl in Chinese sturgeon (Acipenser sinensis), a basal fish.

The gene family DAZ (deleted in Azoospermia), including boule, dazl and DAZ, performs highly conserved functions in germ cell development and fertility across animal phyla. Differential expression patterns have been demonstrated for the family members in invertebrates and vertebrates including fish. Here, we report the identification of boule and dazl and their expression at both RNA and protein levels in developing and mature gonads of Chinese sturgeon (Acipenser sinensis). Firstly, the isolation of the boule and dazl genes in Chinese sturgeon and the observation of the two genes in coelacanth suggest that dazl originated after the divergence of bony fish from cartilaginous fish but before the emergence of the Actinistia. Quantitative real-time PCR and western blot analyses reveal that boule and dazl RNA and proteins are restricted to the testis and ovary. In situ hybridization and fluorescent immunohistochemistry show that the bisexual mitotic and meiotic germ cell expression of dazl RNA and protein is conserved in vertebrates, while Chinese sturgeon boule RNA and protein exhibit mitotic and meiotic expression in the testis, and also likely display mitotic and meiotic expression in female. Moreover, we directly demonstrate for the first time that sturgeon Balbiani body/mitochondrial cloud disperses in the cytoplasm of early developing oocytes and co-localizes with Dazl to some extent. Finally, urbilaterian boule may also have an ancestral function in oogenesis. Taken together, these results provide useful information on the evolution of DAZ family genes, expression patterns and functions in animal reproduction.

One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification.

BACKGROUND: Bean pod mottle virus (BPMV) is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV. METHODS: A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds. RESULTS: The optimized RT-LAMP parameters including 6 mM MgCl2, 0.8 M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn't be used for detection of BPMV using crude RNA extract from soybean seeds. CONCLUSION: A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus.

Molecular and expression characterization of two somatostatin genes in the Chinese sturgeon, Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNA was synthesized from the hypothalamus of Chinese sturgeon, and the full-length cDNAs of two somatostatin (SS) genes were cloned and sequenced. The first cDNA (AsSS1) encodes a 116-amino acid protein that contains the SS(14) sequence at its C-terminal extremity. AsSS1 shows high identity to that of human and other vertebrates. The second cDNA (AsSS2) encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]-SS(14) at its C-terminal extremity. Both the two SS mRNAs were expressed in brain and pituitary with different mRNA levels. But in peripheral tissues, AsSS2 was more widely distributed than AsSS1. High mRNA levels of AsSS2 were found in liver, kidney and heart, while low mRNA levels of AsSS2 were also detected in ovary. Throughout embryogenesis and early larval development only AsSS2 mRNAs were detected. Furthermore, in the hypothalamus of one to five year-old Chinese sturgeon, AsSS2 but not AsSS1 maintained stable expression. The mRNA distribution suggests that the Chinese sturgeon AsSS2 products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development.

Phylogeography of the threatened tetraploid fish, Schizothorax waltoni, in the Yarlung Tsangpo River on the southern Qinghai-Tibet Plateau: implications for conservation.

The phylogeography of Schizothorax waltoni, an endemic and endangered tetraploid schizothoracine fish in the Yarlung Tsangpo River (YLTR) on southern margin of Qinghai-Tibet Plateau (QTP), was investigated using two mitochondrial DNA regions and eleven microsatellite loci. Analyses of concatenated sequences of cytochrome b (1141 bp) and the control region (712 bp) revealed high haplotype diversity and moderate nucleotide diversity. High genetic diversity was observed based on microsatellite variation. Both mtDNA and microsatellite analyses revealed significant genetic differentiation between the eastern population (Mainling) and the other four populations to the west, and non-significant genetic differentiation amongst the three central populations in the west. Significant genetic differentiation was observed between the western population (Shigatse) and the three central populations based on microsatellite analyses alone. Bayesian skyline plot analyses showed that S. waltoni experienced a pronounced population expansion 0.05 to 0.10 Ma. Hierarchical structure analyses of microsatellite data indicated that S. waltoni could be split into three groups (western, central and eastern YLTR). The results indicate that three management units should be considered for S. waltoni. Our findings highlight the need for the conservation and effective management of S. waltoni, which is a key member of the endemic and highly threatened fishes of the QTP.

Phylogeographic patterns and conservation implications of the endangered Chinese giant salamander.

Understanding genetic diversity patterns of endangered species is an important premise for biodiversity conservation. The critically endangered salamander Andrias davidianus, endemic to central and southern mainland in China, has suffered from sharp range and population size declines over the past three decades. However, the levels and patterns of genetic diversity of A. davidianus populations in wild remain poorly understood. Herein, we explore the levels and phylogeographic patterns of genetic diversity of wild-caught A. davidianus using larvae and adult collection with the aid of sequence variation in (a) the mitochondrial DNA (mtDNA) fragments (n = 320 individuals; 33 localities), (b) 19 whole mtDNA genomes, and (c) nuclear recombinase activating gene 2 (RAG2; n = 88 individuals; 19 localities). Phylogenetic analyses based on mtDNA datasets uncovered seven divergent mitochondrial clades (A-G), which likely originated in association with the uplifting of mountains during the Late Miocene, specific habitat requirements, barriers including mountains and drainages and lower dispersal ability. The distributions of clades were geographic partitioned and confined in neighboring regions. Furthermore, we discovered some mountains, rivers, and provinces harbored more than one clades. RAG2 analyses revealed no obvious geographic patterns among the five alleles detected. Our study depicts a relatively intact distribution map of A. davidianus clades in natural species range and provides important knowledge that can be used to improve monitoring programs and develop a conservation strategy for this critically endangered organism.

Comorbidity Characterization Among eMERGE Institutions: A Pilot Evaluation with the Johns Hopkins Adjusted Clinical Groups® System.

Electronic health records (EHR) are valuable to define phenotype selection algorithms used to identify cohorts ofpatients for sequencing or genome wide association studies (GWAS). To date, the electronic medical records and genomics (eMERGE) network institutions have developed and applied such algorithms to identify cohorts with associated DNA samples used to discover new genetic associations. For complex diseases, there are benefits to stratifying cohorts using comorbidities in order to identify their genetic determinants. The objective of this study was to: (a) characterize comorbidities in a range of phenotype-selected cohorts using the Johns Hopkins Adjusted Clinical Groups® (ACG®) System, (b) assess the frequency of important comorbidities in three commonly studied GWAS phenotypes, and (c) compare the comorbidity characterization of cases and controls. Our analysis demonstrates a framework to characterize comorbidities using the ACG system and identified differences in mean chronic condition count among GWAS cases and controls. Thus, we believe there is great potential to use the ACG system to characterize comorbidities among genetic cohorts selected based on EHR phenotypes.

Establishment of intraperitoneal germ cell transplantation for critically endangered Chinese sturgeon Acipenser sinensis.

Recent progress in germ cell transplantation techniques in fish has paved the way for the conservation of endangered species. Here, we developed an intraperitoneal germ cell transplantation procedure using Chinese and Dabry's sturgeon as donor and recipient species, respectively. Histological analysis revealed that primordial germ cells migrated on the peritoneal wall at 16 days post-hatch (dph) in Dabry's sturgeon. The genital ridges of Dabry's sturgeon (recipient) first formed at 28 dph, suggesting that for successful colonization of donor germ cells in the recipient gonads, the transplantation should be performed earlier than this age. Sexual dimorphism of gonadal structure was first observed at 78 dph. Gonadal germ cell proliferation was not seen in either sex during this period. Immunohistochemistry using the anti-Vasa antibody found that donor testes from 2-year-old Dabry's sturgeon mainly consisted of single- or paired-type A spermatogonia, while donor ovaries from 11.5-year-old Chinese sturgeon had perinucleolus stage oocytes and clusters of oogonia. Donor cells isolated from Dabry's sturgeon testes or Chinese sturgeon ovary labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of the 7- or 8-dph Dabry's sturgeon larvae. More than 90% and 70% of transplanted larvae survived after 2 days post-transplantation (dpt) and 51 dpt, respectively. At 51 dpt, PKH26-labeled cells exhibiting germ cell-specific nuclear morphology and diameter were observed in excised recipient gonads by fluorescent and confocal microscopy. The colonization rate of allogeneic testicular germ cell transplantation (Group 1) was 70%, while that of two batches of xenogeneic ovarian germ cell transplantation (Group 2 and Group 3) were 6.7% and 40%, respectively. The ratio of colonized germ cells to endogenous germ cells was 11.96%, 5.35% and 3.56% for Group 1, Group 2 and Group 3, respectively. Thus, we established a germ cell transplantation technique for the critically endangered Chinese sturgeon using the most closely related species as a recipient and demonstrated the successful preparation of transplantable female germ cells from aged adult Chinese sturgeon.

Phylogeography and population genetics of Schizothorax o'connori: strong subdivision in the Yarlung Tsangpo River inferred from mtDNA and microsatellite markers.

The Qinghai-Tibet Plateau (QTP) is a biodiversity hotspot, resulting from its geological history, contemporary environment and isolation. Uplift of the QTP and Quaternary climatic oscillations are hypothesised to have influenced the genetic diversity, population structure and dynamics of all QTP endemic species. In this study, we tested this hypothesis by assaying variation at two mitochondrial DNA regions (cytochrome b and control region) and at 12 microsatellite loci of seven populations of the endemic fish, Schizothorax o'connori from the Yarlung Tsangpo River (YLTR) on the QTP. Analyses revealed one group of six populations to the west, above the Yarlung Tsangpo Grand Canyon (YTGC), and a second group to the east below the YTGC. Estimates of the timing of this east-west split indicate that these groups represent evolutionarily significant units that have evolved separately and rapidly in the middle Pleistocene, at the time of the Kunlun-Huanghe Movement A Phase and the Naynayxungla glaciation. Population dynamic analyses indicate that S. o'connori experienced a pronounced late Pleistocene expansion during the last interglacial period. The results of this study support the hypotheses that the QTP uplift and Quaternary climatic oscillations have played important roles in shaping the population genetics and dynamics of this endemic fish.

[Interspecies diet relationship of Coreius from Yinbin reach of Yangtze River, China].

This study investigated the diet compositions of Coreius heterodon and C. guichenoti in Yibin reach of Yangtze River from April to May, 2012 by carbon and nitrogen stable isotopes technique, and analyzed their diet relationship by the multivariate statistical technique, which covered the diet similarity index, overlap coefficient, and the influence of morphological differences in feeding organ between the two species on diet compositions. The results showed that, 1) the δ13C and δ15N of C. heterodon was from -21.15 per thousand to -20.31 per thousand and 9.67 per thousand to 10.21 per thousand, respectively, which indicated that the fish had an omnivorous possessing carnivorous preference. The δ13C and δ15N of C. guichenoti was from -23.30 per thousand to -21.18 per thousand and 7.40 per thousand to 9.21 per thousand, respectively, which had an omnivorous possessing phytophagous preference. There were some differences in the results between the stable isotopes and the traditional intestinal contents analysis. 2) The diet similarity index and the overlap coefficient was 78.7% and 55.6%, respectively. 3) The principal component analysis (PCA) result showed that the morphological characters of the feeding organs in the two fishes were different. In conclusion, the interspecies competition was not intense between the C. heterodon and C. guichenoti, and as the last investigation of the diet composition and food relationship of the two species before the impoundment of the Xiangjiaba Reservoir, the results could provide reference for analyzing the influence of impoundment on the fish trophic structure in Yibin reach of Yangtze River.

The complete mitogenome sequence of Ptychobarbus dipogon (Cypriniformes: Cyprinidae).

The complete mitochondrial genome of Ptychobarbus dipogon is 16,787 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions: control region (CR) and origin of light-strand replication (OL). The gene order of P. dipogon mitogenome is similar to those observed in most other vertebrates. The complete mitogenome sequence of P. dipogon can provide useful data for further studies on population structure, phylogenetics and conservation genetics of this species.

Characterization of the complete mitochondrial genome of Oxygymnocypris stewartii (Cypriniformes: Cyprinidae).

The complete mitochondrial genome of Oxygymnocypris stewartii is 16,646 bp in size, containing 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (the control region and the origin of light-strand replication). The gene order of O. stewartii mitogenome is similar to those observed in most other vertebrates. The complete mitogenome sequence information of O. stewartii can provide useful data for further studies on phylogenetics, stock evaluation and conservation genetics.

Ovary transcriptome profiling of Coilia nasus during spawning migration stages by Illumina sequencing.

Coilia nasus is an anadromous kind of small to moderate size fish species, and limited transcriptomics research has been performed. In the present study, we performed Illumina sequencing to produce a 22,996,612 clean reads representing with a total of 4,599,079,601 (4.5Gb) nucleotides comprehensive transcript dataset for ovary of C. nasus. Over 20 million total reads were assembled into 63,141 unigenes, and 27,027 annotated genes were predicted by Blastx and ESTScan, respectively. Applying Blast analysis and functional annotation (e.g., GO, COG, Swissprot and KEGG), we have sampled an extensive and diverse expressed gene catalog for C. nasus representing a large proportion of the genes expressed in the ovary development process. This approach will assist in the discovery and annotation of novel genes that play key roles in anadromous fish spawning migration process.

One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.

Molecular and expression analysis of apolipoprotein E gene in the Chinese sturgeon, Acipenser sinensis.

Apolipoproteins are carrier proteins that bind to lipids to form lipoprotein particles and have been shown to play an important role in lipid metabolism. In this study, a full-length cDNA for apolipoprotein E, named AsapoE, was cloned from the Chinese sturgeon (Acipenser sinensis). This cDNA sequence is 1289 bp in length, and codes for a polypeptide of 274 amino acid residues, which is 45% and 42% identical to that of the rainbow trout and zebrafish, respectively, and 39%, 30%, and 29% identical to frog, mouse, and human respectively. The predicted AsApoE protein has a conserved amphipathic α-helix region with the potential to bind to lipids. RT-PCR analysis reveals that AsapoE is expressed in all tissues examined with a preferential expression in the kidney and liver. During the embryo development stage, AsapoE mRNA is low but still detectable at gastrula stage embryos; then AsapoE mRNAs reach a higher level in muscle contraction stage embryos, this relatively stable expression persists during the following embryogenic stages and declines 1 day after hatching. These results will serve as a basis for comparative studies on vertebrate apoE genes.

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals.

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSHbeta (AsFSHbeta) and LHbeta (AsLHbeta) subunit cDNAs are 345, 363, 387 and 414bp in length, and encode mature peptides of 115, 121, 129 and 138aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSHbeta and AsLHbeta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSHbeta and AsLHbeta) in mature sturgeon pituitaries, but only AsFSHbeta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSHbeta was expressed in 4-year-old individuals, whereas in females, AsFSHbeta was just expressed in 5-year-old individuals.

[Reproductive biology and artificial propagation of Acipenser sinensis below Gezhouba Dam].

A total of 36 females and 21 males of Chinese sturgeon Acipenser sinensis were caught in 1998-2004 excluding 2002 to study the characteristics of their reproductive biology and the effect of their artificial propagation. The results showed that the body length (BL), body mass (BM) and age of the females were 240-320 cm, 140-432 kg, and 15-30 years, and those of the males were 153-284 cm, 70-244 kg and 12-26 years, respectively. The inducing rate was 93.1% for females and 100% for males, and the ova had 7 different colors. The absolute fecundity was 200,000-590,000 eggs, with an average of 358,000 eggs, and the relative fecundity to BM was 820-3,020 eggs per kg, with an average of 1,590 eggs per kg. The sperm had 4 different colors. The absolute sperm quantity obtained from one male was 1,000-5,952 ml, with an average of 2,597.8 ml, and the relative sperm quantity to BM was 1.25-31.24 ml . kg(-1), with an average of 13.3 ml . kg(-1). During the study period, the average fertilization rate in artificial propagation was 63.7%, and the hatching rate was 48.1%, with 4,762,000 fry obtained. Compared with the data in 1976, the natural reproductive capacity of the Chinese sturgeon broodstocks declined greatly.

Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi.

Biological production of p-hydroxycinnamic acid (pHCA) from glucose can be achieved via deamination of the aromatic amino acids l-tyrosine or l-phenylalanine. Deamination of l-phenylalanine produces trans-cinnamic acid (CA) which is further hydroxylated in the para position to produce pHCA. However, when tyrosine is used as the substrate, trans-pHCA is produced in one step. This reaction is accomplished by phenylalanine ammonia-lyase (PAL)/tyrosine ammonia-lyase (TAL). Various bacteria and eukaryotic microorganisms were screened for their ability to produce a PAL/TAL enzyme with high TAL activity. Cell-free extracts of the yeast Rhodotorula glutinis possessed the highest level of TAL activity (0.0143U/mg protein) and the lowest PAL/TAL ratio (1.68) amongst species examined. The gene for this enzyme was cloned and expressed in Escherichia coli and the kinetics of the purified PAL/TAL determined. The recombinant PAL/TAL possessed characteristics similar to those of the wild-type enzyme. Functional expression of R. glutinis PAL/TAL enzyme in Saccharomyces cerevisiae cells containing the plant C4H P-450 and P-450 reductase enzymes from Helianthus tuberosus allowed conversion of glucose to pHCA. Addition of l-phenylalanine to these cultures increased pHCA production confirming its production via the PAL route. When R. glutinis PAL/TAL was synthesized in an E. colil-phenylalanine producing strain (ATCC 31882) and grown on glucose, pHCA was formed in the absence of the Cytochrome P-450 and the P-450 reductase enzymes underlining its production via the TAL route without CA intermediacy.