Jian-Qiang Kong

Phenylalanine ammonia-lyase, a key component used for phenylpropanoids production by metabolic engineering

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) catalyzes the deamination of phenylalanine to cinnamate and ammonia, the first step of the phenylpropanoid pathway. PALs are ubiquitous in plants and also commonly found in fungi, but have not yet been detected in animals. Typically, PAL is encoded by a small multigene family and the presence of PAL isoforms is a common observation. PAL belongs to the 3,5-dihydro-5-methylidene-4H-imidazol-4-one-containing ammonia-lyase family and has been shown to exist as a tetramer. Both the forward and reverse reactions catalyzed by PALs were of great interest and have potential industrial and medical applications. This review, therefore, covers the recent developments related to the PAL gene distribution, phenylalanine ammonia-lyase gene family, structure and function study of PALs, as well as several potential applications of PALs. As a key gateway enzyme linking the phenylpropanoid secondary pathway to primary metabolism, PALs were extensively applied in heterologous hosts to produce phenylpropanoids. The review thereby highlights the synthetic potentials of PALs as a key component used in metabolic engineering and synthetic biology. Moreover, the other potential PAL applications, like enzyme replacement therapy of phenylketonuria, as a therapeutic enzyme in cancer treatment and microbial production of L-phenylalanine are also discussed in detail. Together these results provide a synopsis of a more global view of potential applications of PALs than previously available.

Transcriptome-guided discovery and functional characterization of two UDP-sugar 4-epimerase families involved in the biosynthesis of anti-tumor polysaccharides in Ornithogalum caudatum

UDP-glucose 4-epimerase (UGE) and UDP-xylose 4-epimerase (UXE), two important UDP-sugar 4-epimerases, are well known to be essential for de novo biosynthesis of UDP-D-galactose and UDP-L-arabinose, two universal sugar donors for the formation of four galactose- and arabinose-containing anticancer polysaccharides in Ornithogalum caudatum. However, very little is known about their cDNA sequences. Furthermore, the functional significance of the two epimerases in the biosynthesis of these anticancer polysaccharides in O. caudatum had not been documented. Here, we presented a full characterization of UGE and UXE, which were deemed to be responsible for anticancer polysaccharides biosynthesis in O. caudatum. Specifically, a transcriptome-guided search for the two epimerase genes in O. caudatum was first performed in the present study. A total of 4 unigenes sharing high sequence identity with UDP-sugar 4-epimerases were retrieved from transcriptome assembly. Four full-length cDNAs encoding UDP-sugar 4-epimerases, including two UGE-like and two UXE-like genes, were then isolated by reverse transcription polymerase chain reaction (RT-PCR) from O. caudatum. Bio-informatic analysis indicated the two UDP-sugar 4-epimerase families shared two common conserved domains, namely an N-terminal GxxGxxG motif and a catalytic Ser/Thr-Tyr-Lys triad. A phylogenetic analysis revealed the two members in the same UGE family could be classified into two subgroups, revealing their divergently functional significance. These candidate isoenzymes were screened by functional expression in E. coli individually as standalone enzymes. Two UGE-like cDNAs were identified to be bona fide genes, exhibiting both UGE and UXE activities. To further explore the possible role of these epimerase proteins in polysaccharides biosynthesis, transcript profiles of the four genes were subsequently examined by real-time quantitative PCR in various O. caudatum tissues. OcUGE1, OcUGE2 and OcUXE1 were therefore assumed to be responsible for the biosynthesis of the four galactose- and arabinose-containing polysaccharides due to their expression profiles in O. caudatum. Taken together, these data provide further comprehensive knowledge for polysaccharides biosynthesis in O. caudatum and broaden the potential application of UGE in metabolic engineering or synthetic biology as a potential gene part.

Transcriptome-wide identification of sucrose synthase genes in Ornithogalum caudatum

OCAP-2-1, OCAP-2-2, OCAP-3-1 and OCAP-3-3, four glucose-containing polysaccharides from Ornithogalum caudatum, exhibit antitumor activity, suggesting their potential application as natural antitumor drugs. Although the incorporation of glucose into these polysaccharides from UDP-D-glucose is reasonably well understood, the cDNA isolation and functional characterization of genes responsible for UDP-D-glucose biosynthesis from O. caudatum has not been identified. Here, we present a full characterization of the sucrose synthase family, a Leloir glycosyltransferase responsible for UDP-D-glucose biosynthesis from O. caudatum. Specifically, a transcriptome-wide search for Sus genes in O. caudatum was first performed in the present study. A total of 5 unigenes sharing high sequence identity with Sus were retrieved from transcriptome sequencing. Three full-length Sus-like candidates derived from this unigene assembly were then obtained and isolated by reverse transcription polymerase chain reaction (RT-PCR) from O. caudatum. Additional analysis showed two conserved domains (sucrose synthase and glycosyl transferase domains) were present in this family. Phylogenetic analysis indicated that the OcSus1 and OcSus2 could be clustered together into a monocots specific clade, while OcSus3 could be classified into M & D1 category with members from the monocots and dicots species, displaying an evolutionary consistency with other plant species. These candidate isoenzymes were screened by functional expression in E. coli individually as standalone enzymes. All three cDNAs were identified to be bona fide genes and encoded sucrose synthase with varied kinetic properties. To further explore the possible role of these Sus proteins in polysaccharide biosynthesis, transcript profiles of the three genes were subsequently examined by real-time quantitative PCR in various tissues. OcSus1 and OcSus2 were therefore assumed to be responsible for the biosynthesis of the four glucose-containing polysaccharides due to their expression profiles in O. caudatum. Taken together, these data provide further comprehensive knowledge for polysaccharide biosynthesis in O. caudatum and broaden the potential application of Sus in metabolic engineering or synthetic biology as a potential gene part.

Transcriptome-wide identification and characterization of Ornithogalum saundersiae phenylalanine ammonia lyase gene family

OSW-1 is a promising antitumor glycoside present in the Ornithogalum saundersiae plant. Biosynthesis of the p-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically by phenylpropanoid biosynthetic pathway, but molecular biological characterization of related genes has been insufficient. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24), which catalyzes the deamination of L-phenylalanine to yield trans-cinnamic acid, plays a key role in phenylpropanoid metabolism. Thus, the study on the characterization of the genes involved in the OSW-1 biosynthetic pathway, particularly the well-documented genes such as PAL, is essential to further the understanding of the biosynthesis of OSW-1. Here, transcriptomic sequencing of O. saundersiae was performed to speed up the identification of a large number of genes related to OSW-1 biosynthesis. De novo assembly of the transcriptome sequence provided 210 733 contigs, 104, 180 unigenes, and four unigenes showing high similarities with PALs. Two full-length cDNAs encoding PALs (OsaPAL2 and OsaPAL62) from O. saundersiae were cloned using sequence information from these four unigenes. The PAL and tyrosine ammonia lyase (TAL) activities of recombinant OsaPAL proteins were unambiguously determined by HPLC with UV and MS detection as well as by NMR spectroscopy. Subsequently, a series of site-directed mutants were generated with the aim of improving enzyme activity and investigating the importance of particular residues in determining substrate selectivity. The results reveal that the Phe-to-His mutants, i.e., OsaPAL2F134H and OsaPAL62F128H, exhibited higher TAL activity than the corresponding wild types, providing direct evidence that the Phe residue is responsible for substrate specificity. Mutagenesis studies also demonstrated that the Thr-to-Ser mutants, i.e., OsaPAL2T196S and OsaPAL62T194S, showed significantly higher substrate affinity than the wild types. Furthermore, the Gly-to-Ala mutants, i.e., OsaPAL2G209A and OsaPAL62G207A, showed higher PAL and TAL activities. These findings provide further insight into the genes responsible for OSW-1 biosynthesis and will facilitate the future application of OsaPALs in synthetic biology.

Artemisinic acid: A promising molecule potentially suitable for the semi-synthesis of artemisinin

Artemisinic acid, an amorphane sesquiterpene, is isolated from Artemisia annua L. Although having less efficacy than artemisinin, artemisinic acid has a variety of pharmacological activity, such as antimalarial activity, anti-tumor activity, antipyretic effect, antibacterial activity, allelopathy effect and anti-adipogenesis effect. This development has drastically increased artemisinic acid demand worldwide. Although many approaches, namely extraction of artemisinic acid from A.annua L, in vitro production of artemisinic acid by cell and tissue culture, total chemical synthesis and fermentation production by use of synthetic biology technology can improve artemisinic acid production, A.annua L. is currently the only commercial source for the artemisinic acid supply in the international market. Recently tremendous advances, however, demonstrate that the production of artemisinic acid in microorganisms and further semi-synthesis to artemisinin is a feasible complementary strategy that would help reduce artemisinin cost in the future. The key genes encoding for enzymes regulating the biosynthesis of artemisnic acid in planta are fully understood to enable metabolic engineering of the pathway, and results from pilot genetic engineering studies in microbial strains thus far are very inspiring. This review, therefore, covers the recent developments related to the physico-chemical properties of artemisinic acid, bioactivity of this important molecular, solvent extraction strategies and chemical analysis, and highlights a scale production of artemisinic acid by synthetic biology and the relevant enzymes and genes. In the end the status of artemisinic acid in the biosynthesis pathway of artemisinin is discussed in detail. Together these results provide a synopsis of a more global view of artemisinic acid than previously available.

Isolation and characterization of a multifunctional flavonoid glycosyltransferase from Ornithogalum caudatum with glycosidase activity

Glycosyltransferases (GTs) are bidirectional biocatalysts catalyzing the glycosylation of diverse molecules. However, the extensive applications of GTs in glycosides formation are limited due to their requirements of expensive nucleotide diphosphate (NDP)-sugars or NDP as the substrates. Here, in an effort to characterize flexible GTs for glycodiversification of natural products, we isolated a cDNA, designated as OcUGT1 from Ornithogalum caudatum, which encoded a flavonoid GT that was able to catalyze the trans-glycosylation reactions, allowing the formation of glycosides without the additions of NDP-sugars or NDP. In addition, OcUGT1 was observed to exhibit additional five types of functions, including classical sugar transfer reaction and three reversible reactions namely NDP-sugar synthesis, sugars exchange and aglycons exchange reactions, as well as enzymatic hydrolysis reaction, suggesting OcUGT1 displays both glycosyltransferase and glycosidase activities. Expression profiles revealed that the expression of OcUGT1 was development-dependent and affected by environmental factors. The unusual multifunctionality of OcUGT1 broadens the applicability of OcUGT1, thereby generating diverse carbohydrate-containing structures.

cDNA Isolation and Functional Characterization of UDP-d-glucuronic Acid 4-Epimerase Family from Ornithogalum caudatum

d-Galacturonic acid (GalA) is an important component of GalA-containing polysaccharides in Ornithogalum caudatum. The incorporation of GalA into these polysaccharides from UDP-d-galacturonic acid (UDP-GalA) was reasonably known. However, the cDNAs involved in the biosynthesis of UDP-GalA were still unknown. In the present investigation, one candidate UDP-d-glucuronic acid 4-epimerase (UGlcAE) family with three members was isolated from O. caudatum based on RNA-Seq data. Bioinformatics analyses indicated all of the three isoforms, designated as OcUGlcAE1~3, were members of short-chain dehydrogenases/reductases (SDRs) and shared two conserved motifs. The three full-length cDNAs were then transformed to Pichia pastoris GS115 for heterologous expression. Data revealed both the supernatant and microsomal fractions from the recombinant P. pastoris expressing OcUGlcAE3 can interconvert UDP-GalA and UDP-d-glucuronic acid (UDP-GlcA), while the other two OcUGlcAEs had no activity on UDP-GlcA and UDP-GalA. Furthermore, expression analyses of the three epimerases in varied tissues of O. caudatum were performed by real-time quantitative PCR (RT-qPCR). Results indicated OcUGlcAE3, together with the other two OcUGlcAE-like genes, was root-specific, displaying highest expression in roots. OcUGlcAE3 was UDP-d-glucuronic acid 4-epimerase and thus deemed to be involved in the biosynthesis of root polysaccharides. Moreover, OcUGlcAE3 was proposed to be environmentally induced.

Biosynthesis of 7,8-dihydroxyflavone glycosides via OcUGT1-catalyzed glycosylation and transglycosylation

Abstract Herein, a flavonoid glycosyltransferase (GT) OcUGT1 was determined to be able to attack C-8 position of 7,8-dihydroxyflavone (7,8-DHF) via both glycosylation and transglycosylation reactions. OcUGT1-catalyzed glycosylation of 7,8-DHF resulted in the formation of two monoglycosides 7-O-β-D-glucosyl-8-hydroxyflavone (1a), 7-hydroxy-8-O-β-D-glucosylflavone (1b), as well as one diglycoside 7,8-di-O-β-D-glucosylflavone (1c). Under the action of OcUGT1, inter-molecular trans-glycosylations from aryl β-glycosides to 7,8-DHF to form monoglycosides 1a and 1b were observable.

Transcriptome-Wide Identification of an Aurone Glycosyltransferase with Glycosidase Activity from Ornithogalum saundersiae

Aurone glycosides display a variety of biological activities. However, reports about glycosyltransferases (GTs) responsible for aurones glycosylation are limited. Here, the transcriptome-wide discovery and identification of an aurone glycosyltransferase with glycosidase activity is reported. Specifically, a complementary DNA (cDNA), designated as OsUGT1, was isolated from the plant Ornithogalum saundersiae based on transcriptome mining. Conserved domain (CD)-search speculated OsUGT1 as a flavonoid GT. Phylogenetically, OsUGT1 is clustered as the same phylogenetic group with a putative 5,6-dihydroxyindoline-2-carboxylic acid (cyclo-DOPA) 5-O-glucosyltransferase, suggesting OsUGT1 may be an aurone glycosyltransferase. The purified OsUGT1 was therefore used as a biocatalyst to incubate with the representative aurone sulfuretin. In vitro enzymatic analyses showed that OsUGT1 was able to catalyze sulfuretin to form corresponding monoglycosides, suggesting OsUGT1 was indeed an aurone glycosyltransferase. OsUGT1 was observed to be a flavonoid GT, specific for flavonoid substrates. Moreover, OsUGT1 was demonstrated to display transglucosylation activity, transferring glucosyl group to sulfuretin via o-Nitrophenyl-β-d-glucopyranoside (oNP-β-Glc)-dependent fashion. In addition, OsUGT1-catalyzed hydrolysis was observed. This multifunctionality of OcUGT1 will broaden the application of OcUGT1 in glycosylation of aurones and other flavonoids.