Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Abstract

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 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.

Keywords: Annotation; Copy number; Fermentation; Genome; Genome resequencing; Hydrolysate; Plasmid; RNA-Seq; Zymomonas mobilis.

Fig. 1

Map of four plasmids in ZM4 and 2032: pZM32 (a), pZM33 (b), pZM36 (c), and pZM39 (d). Seven different functional groups of encoding protein are indicated in different colors

Fig. 2

Map of xylAB′ duplicated region in the chromosome of Z. mobilis 2032. Pgap_xylA-xylB-yiaB′-yiaA′-wecH′-tetA (genes are in green color and Pgap is in blue color) was inserted into ZMO1237 (ldhA) in 2032. The orange rectangle indicates downstream (DS) region of ldhA and purple arrow indicates either 5′ or 3′ of ldhA. Duplicated Pgap_xylA xylB′ and portion of 3′ end of ldhA and its downstream region is indicated as a line above

Fig. 3

Plasmid copy numbers in Z. mobilis ZM4, 2032, and 8b under anaerobic conditions (a), aerobic conditions (b), and shift from aerobic to anaerobic conditions (c). All Z. mobilis strains were grown in RMG medium under anaerobic, aerobic or shift from aerobic-to-anaerobic conditions, and plasmid copy numbers were measured at both Mid-log and Stationary phases relative to the chromosome terminus assigned as 1 and corrected for PCR efficiency (see “Methods”). The chromosome ori copy number was determined as a control. Plasmid copy number measurements were based on at least three biological replicates, with the standard deviation indicated as error bar

Fig. 4

t-SNE analysis of the plasmid-restricted expression data from the 3 laboratories. Blue, NREL, fermentor with biomass hydrolysates; black, NREL, flasks with rich RMG medium; light gray, GLBRC, 6% ACSH; Orange, GLBRC, 9% ACSH; light green, Univ. Athens (UA), anaerobic; dark green, UA, aerobic. Circles, exponential growth stage (exponential in UA dataset and Glucose stage, Glu, in GLBRC dataset); triangles, later growth stage (stationary stage in UA dataset and xylose stage, Xyl, in GLBRC dataset)

Fig. 5

Comparative fermentation of Z. mobilis 2032 (a) and 8b (b) in 6% ACSH. Left axis: glucose (circle), xylose (square), and ethanol (triangle) concentration in the bioreactors (g/L). Right axis: OD_600 nm for cell growth (diamond)

Fig. 6

Comparative fermentation of Z. mobilis 2032 (a) and 8b (b) in 9% ACSH. Left axis: glucose (circle), xylose (square), and ethanol (triangle) concentration in the bioreactors (g/L). Right axis: OD_600 nm for cell growth (diamond)