Complete genome sequenc e and the expression patter n of plasmids of the model ethanologen Zymomonas mobilis ZM 4 and its xylose-utilizing derivativ es 8 b and 2032

Background: Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2–8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. Results: In this study, we determined the complete chromosome and plasmid sequences of ZM4 and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1–2 © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Open Access Biotechnology for Biofuels *Correspondence: Shihui.Yang@hubu.edu. cn; kmpappas@biol.uoa. gr; yzhang8@wisc.edu Shihui Yang, Jessica M. Vera and Yaoping Zhang contributed equally to this work 1 Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Environmental Microbial Technology Center of Hubei Province, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, China 3 DOE-Great Lakes Bioenergy Research Center (GLBRC), University of Wisconsin-Madison, Madison, WI, USA 4 Department of Genetics & Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens (NKUA), Panepistimiopolis, 15701 Athens, Greece Full list of author information is available at the end of the article Page 2 of 20 Yang et al. Biotechnol Biofuels (2018) 11:125 Background Advances in next-generation sequencing (NGS) technology, mass spectrometry, and integrative omics approaches have facilitated more detailed studies of microbial responses to environmental and stress conditions. However, integrative analysis of multi-omic datasets requires the availability of complete genome sequences, i.e., chromosomal and extrachromosomal genomic content, in addition to accurate gene identification and functional annotation. Zymomonas mobilis is a natural ethanologen being developed for industrial conversion of biomass feedstocks into biofuels [1, 2]. Unlike the classical ethanologen Saccharomyces cerevisiae, which uses the Embden–Meyerhof–Parnas (EMP) pathway for glycolysis, Z. mobilis uses the Entner–Doudoroff (ED) pathway, resulting in less ATP produced per molecule of glucose consumed and higher ethanol yield [3]. Furthermore, Z. mobilis is tolerant to many inhibitors found in biomass hydrolysates and therefore has the potential to be developed as a platform catalyst using feedstocks from widely different sources [4–6]. Recently, it was demonstrated that Z. mobilis can utilize atmospheric N2 as the sole nitrogen source during fermentation without affecting ethanol yield, which could significantly reduce the cost of cellulosic ethanol production [7]. Besides ethanol, Z. mobilis has recently been engineered to produce 2,3-butanediol, which can serve as a precursor for the generation of advanced biofuels and bioplastics [8]. Phenotype microarray profiling of Z. mobilis ZM4 indicates that Z. mobilis is closely related to yeast physiologically [9]. Following the first transcriptomic and metabolomic study of Z. mobilis during aerobic and anaerobic fermentation [10], transcriptomics has been widely used to study the stress responses of Z. mobilis to high glucose concentration, ethanol, furfural, phenolic compounds, and other inhibitors [11–14]. In addition, proteomics studies have provided an in-depth understanding of acetate and ethanol stress responses [15, 16]. With the rapidly decreasing costs of next-generation sequencing (NGS), it is expected that NGS-based systems biology studies will be widely applied in the future. However, despite advances in this direction, improvements in genome sequencing accuracy and annotation are still required to meaningfully interpret microbial physiology via high-throughput omics analyses. To facilitate multi-omic data analyses, the genomes of eight Z. mobilis strains have been completely sequenced, including model strain ZM4 [17–23]. In 2009, we improved the genome annotation of Z. mobilis ZM4 (AE008692.2) [24]. We additionally reported the sequence and annotation of five plasmids in ZM4, which were absent in the original ZM4 genome sequence report in 2005 [17]. These plasmids, pZZM401 to pZZM405, were deposited in 2010 under the GenBank Acc. No. CP001881.1–CP001885.1. However, due to a strain identity issue propagated by scientific literature, patent deposits, and bacterial culture collections in both Europe and USA [20, 25], these plasmid sequences were actually derived from strain CP4 instead of the previously reported ZM4. This issue became apparent when (1) CP4 was sequenced to completion in 2013 and proved not to be identical to ZM4 as previously thought [20]; (2) an already sequenced ZM4 41-kb plasmid (plasmid 1; AY057845.1) was not identical to any reported ZM4 or CP4 plasmids, instead each and every newly sequenced CP4 plasmid has high similarity to plasmids from pZZM401 to pZZM405; and (3) NGS reads of ZM4 from both genome resequencing and RNA-Seq studies were unable to map to the majority of the pZZM401 to pZZM405 plasmid sequences. Because of these discrepancies, we decided to resequence Z. mobilis ZM4 as well as two engineered xylose-utilizing derivatives 2032 and 8b that are implemented in lignocellulose fermentations. Our foremost aim was to identify and annotate missing ZM4 plasmids and assign potential physiological roles for plasmid genes by evaluating gene expression in a variety of growth conditions and lignocellulosic hydrolysates. Plasmid copy numbers were also examined under different conditions. Results and discussion Genome sequences of Z. mobilis ZM4 and its xylose‐utilizing derivatives 2032 and 8b Identification of four plasmids in ZM4 and its derivatives To re-examine the genome of the Z. mobilis model strain ZM4 and obtain an unequivocal view of its plasmids, we per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. Conclusions: The complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.