Zymomonas diversity and potential for biofuel production

Abstract

Background Zymomonas mobilis is an aerotolerant α-proteobacterium, which has been genetically engineered for industrial purposes for decades. However, a comprehensive comparison of existing strains on the genomic level in conjunction with phenotype analysis has yet to be carried out. We here performed whole-genome comparison of 17 strains including nine that were sequenced in this study. We then compared 15 available Zymomonas strains for their natural abilities to perform under conditions relevant to biofuel synthesis. We tested their growth in anaerobic rich media, as well as growth, ethanol production and xylose utilization in lignocellulosic hydrolysate. We additionally compared their tolerance to isobutanol, flocculation characteristics, and ability to uptake foreign DNA by electroporation and conjugation. Results Using clustering based on 99% average nucleotide identity (ANI), we classified 12 strains into four clusters based on sequence similarity, while five strains did not cluster with any other strain. Strains belonging to the same 99% ANI cluster showed similar performance while significant variation was observed between the clusters. Overall, conjugation and electroporation efficiencies were poor across all strains, which was consistent with our finding of coding potential for several DNA defense mechanisms, such as CRISPR and restriction–modification systems, across all genomes. We found that strain ATCC31821 (ZM4) had a more diverse plasmid profile than other strains, possibly leading to the unique phenotypes observed for this strain. ZM4 also showed the highest growth of any strain in both laboratory media and lignocellulosic hydrolysate and was among the top 3 strains for isobutanol tolerance and electroporation and conjugation efficiency. Conclusions Our findings suggest that strain ZM4 has a unique combination of genetic and phenotypic traits that are beneficial for biofuel production and propose investing future efforts in further engineering of ZM4 for industrial purposes rather than exploring new Zymomonas isolates.

Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

“…Although Z. mobilis could be an ideal host for lignocellulosic bioproducts, it is disadvantageous for bioethanol production using grains since it does not have enzymes such as amylase and maltase to utilize sugars other than sucrose, glucose, and fructose (Xia et al 2019 ). It seems that the high sugar environment of fruit saps that Z. mobilis evolved to thrive in shaped its capability of utilizing sugars of sucrose, glucose, and fructose that usually exist in fruits, and the unique features of its hopanoid membrane structure and anaerobic Entner–Doudoroff (ED) pathway with efficient enzymes of pyruvate decarboxylase (Pdc) and alcohol dehydrogenases (Adhs) help it tolerate and efficiently utilize high concentration sugars for high ethanol production and tolerance (Brenac et al 2019 ; Felczak et al 2021 ; Todhanakasem et al 2020 ; Wang et al 2018 ; Yang et al 2021 ) (Additional file 1 : Fig. S1).…”

Zymomonas mobilis ZM4 Utilizes an NADP + -Dependent Acetaldehyde Dehydrogenase To Produce Acetate

“…However, there is no acetaldehyde dehydrogenase annotated in the Z. mobilis Z6 genome ( 15 ). Further, we have not found an annotated acetaldehyde dehydrogenase in any other Z. mobilis genome ( 7 ). As a result, most genome scale metabolic models for Z. mobilis only include acetate as a by-product of less central reactions, such as amino acid biosynthesis ( 10 , 16 , 17 ).…”

“…Stressors present in lignocellulosic hydrolysates may also lead to by-product formation. We previously observed that Z. mobilis produces 5 to 10 mM acetate when grown in anaerobic lignocellulosic hydrolysate ( 7 ). Acetate production during lignocellulose fermentation is problematic because it reduces the efficiency of biofuel production and further increases the acetate concentration.…”

“…Here, we refer to the locus ZMO1754 as aldB due to the functional similarity to the E. coli gene of the same name. The ZM4 strain was used because it is the most promising strain for hydrolysate fermentation ( 7 ), and recently developed genome modification tools facilitated generation of a markerless deletion strain in this background ( 19 ).…”

The Delignification of Plants Residue Substrate and Accelerated Fungal Consortium Growth-Saccharification: A Practical Approach

“…Therefore, the decreasing sugar concentration at 10% Brix initial with all fermentation process factors needs enhancement for the larger scale. Further research is required to distinguish xylose hydrolysate in the bioethanol process, as Felczak et al (2021) reported the incapability limit of its role in ethanol fermentation.…”