Yang Shihui, Vera Jessica M, Grass Jeff, Savvakis Giannis, Moskvin Oleg V, Yang Yongfu, McIlwain Sean J, Lyu Yucai, Zinonos Irene, Hebert Alexander S, Coon Joshua J, Bates Donna M, Sato Trey K, Brown Steven D, Himmel Michael E, Zhang Min, Landick Robert, Pappas Katherine M, Zhang Yaoping
1Hubei 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.
2DOE-National Bioenergy Center, National Renewable Energy Laboratory (NREL), Golden, CO 80401 USA.
Biotechnol Biofuels. 2018 May 2;11:125. doi: 10.1186/s13068-018-1116-x. eCollection 2018.
is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in 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.
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 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion.
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.
是一种正在被开发并用作工业生物燃料生产者的天然产乙醇菌。迄今为止，已有8个菌株完成了全基因组测序，发现它们含有2至8个天然质粒。然而，此前尚未对预测的质粒基因及其对细胞适应性的贡献进行系统验证。此外，模式菌株ZM4中质粒的确切数量和身份尚不清楚。ZM4中质粒基因功能信息的缺乏阻碍了对这种模式生物燃料生产菌株的持续研究。
在本研究中，我们测定了ZM4及其工程化木糖利用衍生物2032和8b的完整染色体和质粒序列。与之前发表并修订的ZM4染色体序列相比，此处报道的ZM4染色体序列包含65个核苷酸序列变异以及一个2400 bp的插入片段。在所有三个菌株中均鉴定出四个质粒，由于插入了用于扩大底物利用的异源DNA，在菌株ZM4和2032中预测有150个质粒基因，在菌株8b中预测有153个质粒基因。然后使用Blast2GO、InterProScan和系统生物学数据分析对质粒基因进行注释，发现大多数基因在其他生物体中有明显的直系同源物或可识别的保守结构域。为了验证质粒基因预测，使用RNA-Seq来绘制转录本图谱，并比较在各种生长条件下的相对基因表达，包括厌氧和好氧条件，或在不同浓度的生物质水解产物中的生长情况。总体而言，质粒基因对不同水解产物浓度的反应比对氧气可用性的反应更敏感。此外，我们的结果表明，尽管所有质粒的拷贝数都很低（每个细胞约1至2个），但某些质粒的拷贝数在特定生长条件下或由于异源基因插入而有所变化。
本研究报道了ZM4和两个木糖利用衍生物的完整基因组，重点是鉴定和表征质粒基因。首次报道了质粒基因的注释、验证、在感兴趣的生长条件下的表达水平以及对宿主适应性的贡献。
