Expression and functional analysis of the lysine decarboxylase and copper amine oxidase genes from the endophytic fungus Colletotrichum gloeosporioides ES026

Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer’s disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.

some endophytic fungi associated with H. serrata are capable of producing HupA [12][13][14] with Colletotrichum gloeosporioides ES026 yielding 45 μg/g dried mycelium according to our previous study 15 . However, HupA production by these endophytes is hindered by low yields and the loss of biosynthetic capability after several generations. Therefore, methods involving overexpression of the enzymes associated with HupA biosynthesis need to be developed in a heterologous host if stable and efficient production is to be achieved [15][16][17] .
Although HupA biosynthesis remains poorly understood, previous studies revealed its initiation by the decarboxylation of L-lysine to generate cadaverine, with the subsequent formation of 5-aminopentanal. Conversion of L-lysine to cadaverine and cadaverine to 5-aminopentanal is catalyzed by lysine decarboxylase (LDC) and copper amine oxidase (CAO), respectively [18][19][20] . LDC and CAO were annotated and confirmed as the first enzymes known to participate in HupA biosynthesis in H. serrata 17 , and de novo RNA sequencing of C. gloeosporioides ES026 performed by Zhang et al. 21 confirmed their roles in the HupA biosynthetic pathway. LDC and CAO are found in both eukaryotes and prokaryotes, including plants, mammals, bacteria, yeast, and fungi. In this study, we established a genetic expression system in C. gloeosporioides capable of high degrees of stable HupA expression. Additionally, we successfully cloned and expressed the CgLDC and CgCAO genes in Escherichia coli and identified the activities of CgLDC and CgCAO associated with HupA biosynthesis. Our results provide valuable insights into genetic modification of strains for selective overexpression of biosynthetic enzymes.

Prokaryotic expression of CgLDC and CgCAO and protein purification. cDNA fragments of CgLDC
(769 bp) and CgCAO (2072 bp) were obtained and cloned from C. gloeosporioides ES026 into the pET28a vector to yield the expression plasmids pET28a-CgLDC and pET28a-CgCAO (Fig. 1). The recombinant proteins CgLDC and CgCAO with hexahistidine-tags at their respective C-terminal regions were expressed in E. coli BL21 (DE3) cells and purified to homogeneity using Ni-affinity chromatography. The purified proteins migrated as a single band according to SDS-PAGE analysis in agreement with predicted molecular masses of 28 kDa and 76 kDa, respectively (Fig. 2).
In vitro enzyme assays. LC-MS analysis revealed that CgLDC catalyzed the conversion of L-lysine to cadaverine, and CgCAO converted cadaverine to 5-aminopentanal (Fig. 3), which is the putative biosynthetic precursor of HupA (Fig. 4). By contrast, no catalytic activity was detected from the inactive forms of CgLDC or CgCAO. These results suggested possible CgLDC and CgCAO involvement in HupA biosynthesis.

Transformation of C. gloeosporioides ES026 and qRT-PCR analysis.
To validate the relationship between CgLDC and CgCAO expression and HupA production, 10 CgLDC-and CgCAO-overexpressing plasmids containing different promoters were constructed (Fig. 5). According to methods used for Agrobacterium transformation, C. gloeosporioides ES026 was transformed using the 10 plasmids, and a randomly selected transformant was confirmed by PCR. Our results indicated amplification of appropriately sized DNA fragments (769 bp and 2072 bp, Fig. 6), verifying C. gloeosporioides ES026 genetic transformation. Quantification by qRT-PCR of CgLDC and CgCAO expression during fermentation indicated that the PagdA-CgLDA and PalcA-CgCAO transformants exhibited the highest expression levels (Fig. 7).
Measurement of HupA production. To investigate transformant effects on HupA production, HupA yield associated with all mutants was analyzed by LC-HRMS. Our results showed that different expression levels of CgLDC and CgCAO produced different HupA yields; however, high levels of CgLDC and CgCAO expression resulted in higher yields of HupA, although transformants exhibiting the highest expression levels did not produce the highest yields of HupA. Two genetically altered strains (Polic-CgLDC and PgpdA-CgCAO) yielded stable, high-yielding HupA production (Fig. 8). Our findings revealed that CgLDC and CgCAO were involved in HupA biosynthesis, but that the HupA-synthesis pathway was regulated by separate enzymes.

Discussion
AD affects millions of people worldwide and is among the four principal death-causing diseases, including heart disease, cancer, and stroke. HupA isolated from H. serrata is a natural acetylcholinesterase inhibitor used to treat AD. As mentioned in the introduction, very few biosynthetic studies have been performed with HupA, although no investigations have been reported that have attempted to identify the biosynthetic pathway leading directly to HupA, two enzymes (LDC and CAO) have been proposed as the entry point enzymes into the pathway to the HupA 22, 23 . However, work on that enzymes have only been performed in nonrelated taxa 24 . Nevertheless, the feeding that catalyze key transformations in the biosynthesis of HupA and other Lycopodium alkaloids. In this study, next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 was performed, and  22,25,26 , the HupA biosynthetic pathway was investigated. HupA biosynthesis involves primary and secondary enzyme conversion, initiating with acetyl-CoA and biotin and ending with the development of L-lysine, followed by secondary metabolism involving the development of cadaverine. LDC converts L-lysine to cadaverine, and CAO converts cadaverine to 5-aminopentanal and piperideine. Sun et al. 27 cloned CAO genes from H. serrata, and Du Zhu et al. 28 cloned LDC genes into the endophytic fungus Shiraia sp. Slf14 from H. serrata, enabling verification of specific characteristics related to LDC and CAO biosynthesis of lycopodium alkaloids. Pelletierine, which is a precursor, is also converted, resulting in HupA synthesis. HupA biosynthesis involves LDC as the first enzyme and CAO as the second enzyme, with LDC    transforming L-lysine to cadaverine, and CAO transforming cadaverine to 5-aminopentanal in lycopodium alkaloid biosynthesis. According to Kyoto Encyclopedia of Genes and Genomes analysis, there is only one pathway involved in synthesizing 5-aminopentanal catalyzed by LDC and CAO.
Recombinant plasmids using different promoters to overexpress CgLDC and CgCAO in C. gloeosporioides ES026 were constructed, and their expression was determined by qRT-PCR. Additionally, the differential expression of key enzymes involved in HupA biosynthesis and associated metabolic pathways were analyzed, with results indicating that elevated expression of CgLDC and CgCAO produced increased levels of HupA as compared with wild-type C. gloeosporioides ES026.
In this study, according to the C. gloeosporioides ES026 genome analysis, CgLDC and CgCAO from C. gloeosporioides ES026 are unique genes, were investigated for their conversion of L-lysine to 5-aminopentanal in HupA biosynthesis. CgCAO is different from HsCAO 18 , which can produce 5-aminopentanal. Our results indicated that these enzymes could be efficiently expressed in C. gloeosporioides, that the resulting CgLDC was capable of cadaverine production, and the resulting CgCAO was capable of 5-aminopentanal production, both of which are HupA biosynthetic intermediates, but In vitro, this reaction may be weak, experiments are needed to investigate whether CgLDC and CgCAO have similar properties to other LDCs and CAOs. These findings revealed that genetic modification of C. gloeosporioides ES026 resulted in a variant capable of stable, high-yield production of HupA. Furthermore, we observed that transformants yielding the highest expression of LDC and CAO did not produce the highest yields of HupA, which might have been due to interference by other enzymes involving in HupA biosynthesis. Further investigation is required to elucidate additional details regarding the pathways involved in HupA biosynthesis.

Materials and Methods
Fungal strains and plasmids. The strain C. gloeosporioides ES026, which produced the highest amount of HupA, was isolated from H. serrata and preserved at the China Center for Type Culture Collection (CCTCC No. 2011046; Wuhan, China). E. coli BL21 and DH10B cells were cultured in Luria broth (LB) at 37 °C. The plasmid pET28a containing the kanamycin-resistance gene was used as an assisting plasmid for the transformation of E. coli BL21 cells. Plasmids pGB92, pGB93, pGB94, pGB95, and pGB96 containing the hygromycin B resistance were used as assisting plasmids for the transformation of C. gloeosporioides ES026.

CgLDC and CgCAO expression in E. coli BL21 (DE3) cells and protein purification.
According to the C. gloeosporioides ES026 genome sequence and transcriptome analysis 21 , the coding regions of CgLDC and CgCAO were amplified by polymerase chain reaction (PCR) from C. gloeosporioides ES026 genomic DNA. PCR products were purified using a gel-extraction kit (Omega Bio-tek, Norcross, GA, USA) and cloned into the pET-28a vector between the EcoRI and NdeI restriction sites to create plasmids pET28a-CgLDC and pET28a-CgCAO for production and purification of the target proteins. The plasmids expressed recombinant proteins containing a hexahistidine-tag at the C-terminus. Subsequently, pET28a-LDC and pET28a-CAO were transformed into E. coli BL21 cells via heat shock, and transformants were verified by PCR and restriction-enzyme digestion.
Cells were cultured to an OD 600 of between 0.6 and 0.8 in LB medium containing 100 µg/mL kanamycin at 37 °C and shaking at 200 rpm. Isopropyl β-D-1-thiogalactopyranoside and CuSO 4 were added to the culture medium to a final concentration of 0.1 mM and 50 µM, respectively, to induce the expression of recombinant CgLDC and CgCAO. The induced broth was maintained at 16 °C with shaking at 200 rpm for an additional 16 h. Cells were collected by centrifugation at 4 °C at 5000 g for 5 min, resuspended in buffer A [50 mM Tris-HCl, 300 mM NaCl, and 4 mM 2-mercaptoethanol (pH 7.6)], and lysed by sonication. Lysates were then centrifuged  The CgCAO reaction mixture was prepared according to methods reported by Sun et al. 30 . The reaction contained 1 mM cadaverine and 0.6 mg/mL purified recombinant CgCAO in 50 mM Tris-HCl buffer (pH 8.0) at a final volume of 500 µL. The mixture was incubated at 25 °C for 16 h, and a separate reaction with boiled (inactive) CgCAO was used as the negative control. Reaction products were extracted with methanol and separated and analyzed by LC-MS.
Transformation of C. gloeosporioides ES026. Agrobacterium-mediated transformations were performed according to the methods of Li et al. and Gong et al. 31,32 , with some modifications. Bacterial cultures were diluted to OD 600 = 0.3 using induction medium (IM) containing 200 mM acetosyringone (AS) and were mixed 1:1 with a conidial suspension (10 6 spores mL −1 ) spread over glass paper on a Co-IM plate containing 400 mM AS. After co-cultivation at 25 °C for 36 h, the membranes were removed, inverted, and placed mycelia-side down onto potato dextrose agar (PDA) plates containing 200 μg/mL cephalosporin to counter-select bacteria and 200 μg/mL hygromycin-B to select for C. gloeosporioides transformants. After incubation at 25 °C for 3 to 5 days, transformed colonies were transferred to PDA plates for the second round of selection. Each transformant just transferred one recombinant plasmid. The transformants were verified by genomic DNA extraction and PCR identification.

RNA isolation and quantitative real-time reverse transcription PCR (qRT-PCR) analysis. Total
RNA was extracted from 1 g mycelia from C. gloeosporioides ES026 cultured for 5 days using the RNeasy plant mini kit (Qiagen, Hilden, Germany) according to manufacturer instructions. A PrimeScript reagent kit with gDNA Eraser (TaKaRa Clontech, Shiga, Japan) was used for cDNA synthesis according to manufacturer's protocol. qRT-PCR was performed in 96-well plates using the StepOne Plus real-time PCR system (Applied Biosystems, Darmstadt, Germany). Each reaction mixture consisted of 12.5 µL 2× SYBR Taq mix (TaKaRa Clontech), 1 µL of forward and reverse primers (10 pmol each), 25 ng of the cDNA, and diethylpyrocarbonate water to a final volume of 25 µL. The oligonucleotide PCR primer pairs are listed in Table 1. The cycling program involved an initial cycle at 94 °C for 30 s, followed by 40 cycles of denaturation at 94 °C for 5 s and annealing and extension at 60 °C for 50 s. mRNA expression levels of the target genes were normalized to the mRNA expression level of the reference gene Tubalin. Comparative expression levels were measured by the 2 −ΔΔCT technique using StepOne version 2.3 and DataAssist software (Applied Biosystems). Measurement of HupA production. Transfer of transformant solution was performed according to the method of Zhao et al. 33,34 with minor modifications. Fermented mycelia were collected by centrifugation at 12,000 g or 10 min, followed by drying at 40 °C overnight and grounding into powder. For chemical extraction, each sample of raw material (1 g) was produced using 0.5% HCl [(30 mL (w/v)] overnight, followed by ultrasonication in a water bath at 40 °C for 1 h. The ingredients were then filtered, and the filtrates were rendered with ammonia solution to pH 9.0. After 1 h, the water phase was extracted three times with chloroform, and the combined chloroform extracts were evaporated to dryness in vacuo. The dry residue was mixed with 1 mL methanol, passed through a 0.45-µm polytetrafluoroethylene syringe filter, and analyzed by LC-HRMS (Agilent Zorbax SB-C18; 150 mm × 4.6 mm, 5-µm diameter, operation of the mass spectrometer (MS) was in electrospray positive ion mode. The MS source and chamber conditions were optimised to give maximum analyte signal intensity as follows: Spray voltage: +3500 V; Capillary Temperature: 320 °C; Sheath Gas: 30.0 psi; Aux Gas: 5.0 psi. Probe Heater Temperature: 300 °C; Scan Range: 50-600 m/z; Scan Rate: 1 Hz). The mobile phases consisted of H 2 O and 5% acetonitrile or 100% acetonitrile (65%: 35%) at a flow rate of 0.6 mL/min. Quantification was performed using the standard curve generated from the HupA standard over a concentration range of between 0.5 and 8.0 mg/L, where the peak area and height showed linear correlations with the absorbance (R 2 = 0.9991).