marine drugs Communication Full-Length Transcriptome of Thalassiosira weissflogii as a Reference Resource and Mining of Chitin-Related Genes Haomiao Cheng 1,2,3, Chris Bowler 4, Xiaohui Xing 5,6,7, Vincent Bulone 5,6,7, Zhanru Shao 1,2,* and Delin Duan 1,2,8,* 1 CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; [email protected] 2 Laboratory for Marine Biology and Biotechnology, Pilot Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China 3 University of Chinese Academy of Sciences, Beijing 100049, China 4 Institut de Biologie de l’ENS (IBENS), Département de Biologie, École Normale Supérieure, CNRS, INSERM, Université PSL, 75005 Paris, France; [email protected] 5 Division of Glycoscience, Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology (KTH), AlbaNova University Centre, 10691 Stockholm, Sweden; [email protected] (X.X.); [email protected] (V.B.) 6 Australian Research Council Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae 5064, Australia 7 Adelaide Glycomics, School of Agriculture Food and Wine, University of Adelaide, Waite Campus, Urrbrae 5064, Australia 8 State Key Laboratory of Bioactive Seaweed Substances, Qingdao Bright Moon Seaweed Group Co., Ltd., Qingdao 266400, China * Correspondence: [email protected] (Z.S.); [email protected] (D.D.) Citation: Cheng, H.; Bowler, C.; Xing, X.; Bulone, V.; Shao, Z.; Duan, D. Abstract: β-Chitin produced by diatoms is expected to have significant economic and ecological Full-Length Transcriptome of value due to its structure, which consists of parallel chains of chitin, its properties and the high Thalassiosira weissflogii as a Reference abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related Resource and Mining of Chitin- Related Genes. Mar. Drugs 2021, 19, genes in diatoms owing to the lack of omics-based information. In this study, we first compared the 392. https://doi.org/10.3390/ chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and md19070392 chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. Academic Editors: Detmer Sipkema, In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs Leila Tirichine and Wim Vyverman (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and Received: 10 June 2021 the complete biosynthetic pathways of chitin and chitosan were explored. The information presented Accepted: 8 July 2021 here will facilitate T. weissflogii molecular research and the exploitation of β-chitin-derived high-value Published: 13 July 2021 enzymes and products. Publisher’s Note: MDPI stays neutral Keywords: PacBio sequencing; full-length transcriptome; Thalassiosira weissflogii; chitin; chitosan with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Chitin, a polymer of 1,4-linked β-D-N-acetylglucosaminyl residues, is the second most abundant natural biopolymer after cellulose, and is widely distributed across taxa [1–3]. Copyright: © 2021 by the authors. N Licensee MDPI, Basel, Switzerland. Together with its partially de- -acetylated derivative chitosan, chitin has many biomedical This article is an open access article applications, such as wound healing, artificial organs and drug delivery [4]. The parallel distributed under the terms and arrangement of chitin chains in the β-chitin allomorph confers specific properties to the conditions of the Creative Commons polymer, such as higher solubility, reactivity and swelling compared to the most abundant Attribution (CC BY) license (https:// form of chitin, α-chitin (Figure1)[ 5,6]. There are very few classes of organisms that produce creativecommons.org/licenses/by/ β-chitin, such as the diatom Thalassiosira sp. [7–9]. Various diatoms have been previously 4.0/). reported to produce chitin, mainly in the genera Thalassiosira and Cyclotella [10,11]. The first Mar. Drugs 2021, 19, 392. https://doi.org/10.3390/md19070392 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, x 2 of 14 Mar. Drugs 2021, 19, 392 2 of 13 been previously reported to produce chitin, mainly in the genera Thalassiosira and Cy‐ reportclotella of[10,11]. the occurrence The first ofreport chitin of in the diatoms occurrence was in ofT. chitin weissflogii in diatoms(fluviatilis was), in which T. weissflogii showed that(fluviatilisT. weissflogii), whichchitin showed represented that T. weissflogii up to 34% chitin of the totalrepresented cell mass up (including to 34% of the the silica) total [ cell12]. Themass content (including and structurethe silica) of [12]. chitin The have content been and widely structure studied of in chitin diatoms, have but been its widely biosynthetic stud‐ pathwayied in diatoms, based but on the its biosynthetic sequencing and pathway gene annotationbased on the is sequencing incomplete [and13,14 gene]. In annotation this study, weis incomplete first measured [13,14]. the chitinIn this content study, in we the first cell measured walls of three the chitin diatom content species, inThalassiosira the cell walls ro- tulaof three, T. pseudonana diatom species,and T.Thalassiosira weissflogii ,rotula and found, T. pseudonana that T. weissflogii and T. weissflogiihad the, and highest found chitin that content.T. weissflogii However, had the apart highest from chitin the identification content. However, of chitinous apart from fibres the [9 identification], our knowledge of chi of‐ chitintinous in fibresT. weissflogii [9], ouris knowledge limited, which of chitin impedes in T. the weissflogii exploitation is limited, of chitin which and chitin-related impedes the enzymesexploitation from of thischitin class and of chitin organisms.‐related enzymes from this class of organisms. Figure 1. Structure ofof α‐α-chitinchitin andand β‐β-chitin.chitin. Anti-parallelAnti‐parallel arrangement of chitin chainschains inin α‐α-chitinchitin whereas parallel arrangement forfor β‐β-chitin.chitin. Diatoms are a major groupgroup ofof phytoplanktonphytoplankton andand contributecontribute approximatelyapproximately 20% ofof global primary primary productivity productivity [15,16]. [15,16]. The The last last two two decades decades have have witnessed witnessed a surge a surge in dia in‐ diatomtom gene gene information, information, with with complete complete genomes genomes [17–19] [17 –and19] a and number a number of transcriptomes of transcrip- tomessequenced sequenced [20–22]. [ 20By– 22contrast,]. By contrast, T. weissflogiiT. weissflogii genes cangenes only be can referred only be to referred the transcripts to the transcriptsreleased by released the Marine by the Marine Microbial Microbial Eukaryote Eukaryote Transcriptome Transcriptome Sequencing Sequencing Project (MMETSP) [[23],23], andand no no full-length full‐length reference reference database database is is openly openly available available so far.so far. Genes Genes en- β codingencoding enzymes enzymes metabolising metabolising high-value high‐value products products such such as -chitinas β‐chitin and theand full the potential full poten of‐ T.tial weissflogii of T. weissflogiiremain toremain be unveiled. to be unveiled. Pacific BioSciences Pacific BioSciences (PacBio) single-molecule(PacBio) single‐ real-timemolecule (SMRT)real‐time sequencing (SMRT) sequencing enables the discoveryenables the of noveldiscovery genes of and novel gene genes product and isoforms. gene product It pro- vides longer reads, making them suitable for biological problems that are poorly solved isoforms. It provides longer reads, making them suitable for biological problems that are by second-generation sequencing (SGS) [24]. A full-length (FL) transcriptome obtained by poorly solved by second‐generation sequencing (SGS) [24]. A full‐length (FL) transcrip‐ PacBio sequencing is an alternative complete transcript assembly for a non-model species. tome obtained by PacBio sequencing is an alternative complete transcript assembly for a In the present study, the FL transcriptome of T. weissflogii was sequenced to elucidate the non‐model species. In the present study, the FL transcriptome of T. weissflogii was se‐ genetic profile and uncover the chitin-related genes of this diatom species. Our study quenced to elucidate the genetic profile and uncover the chitin‐related genes of this dia‐ highlights information for β-chitin metabolism in T. weissflogii, provides a reference genetic tom species. Our study highlights information for β‐chitin metabolism in T. weissflogii, dataset for future studies and assists in evolutionary studies to a broad taxonomy. The FL provides a reference genetic dataset for future studies and assists in evolutionary studies transcriptome will enable the construction of chitin metabolic pathways and facilitate the to a broad taxonomy. The FL transcriptome will enable the construction of chitin meta‐ in vitro application of chitin-related enzymes. bolic pathways and facilitate the in vitro application of chitin‐related enzymes.