Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4

Felczak, Magdalena M.; Jacobson, Tyler B.; Ong, Wai Kit; Amador‐Noguez, Daniel; TerAvest, Michaela A.

doi:10.3389/fmicb.2019.02270

Cited by 13 publications

(8 citation statements)

References 22 publications

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“…A primary focus of this work was to identify RM systems of Z. mobilis ZM4 and to create strains with improved ability to take up plasmids encoding heterologous metabolic functions. Indeed, we improved the conjugation efficiency of plasmids containing genes that encode β-galactosidase, a xylose utilization pathway ( 36 – 38 ), and phosphoglucoisomerase ( 49 , 50 ). Given the utility of β-galactosidase as a widespread reporter in gene expression studies, our development of a strain lacking RM systems provides a new useful tool for analyzing promoter-specific gene expression in Z. mobilis ZM4.…”

Section: Discussionmentioning

confidence: 99%

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

Lal

Wells

Myers

et al. 2021

Appl Environ Microbiol

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Zymomonas mobilis has emerged as a promising candidate for production of high value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr -like Type IV Restriction Modification (RM) system, a Type I-F CRISPR system, a chromosomal Type I RM ( hsdMS c ) and a previously uncharacterized Type I RM system, located on an endogenous plasmid ( hsdRMS p ). The DNA recognition motif of HsdRMS p was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMS c and hsdRMS p systems to the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMS c and HsdRMS p recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction - strains that we constructed, and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. Importance Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study we have developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identified a previously unknown Type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis .

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Section: Discussionmentioning

confidence: 99%

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

Lal

Wells

Myers

et al. 2021

Appl Environ Microbiol

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“…To begin to link the presence of DNA defense systems with physiology, we measured the efficiency of electroporation and conjugation of a plasmid into each of the Z. mobilis strains and normalized the efficiency to that of ZM4 (Table 5 ). We utilized a plasmid (pRL814) that was previously constructed from standard synthetic biology parts for use in Z. mobilis [ 46 , 47 ] . pRL814 carries a spectinomycin resistance cassette and an IPTG-inducible gfp .…”

Section: Resultsmentioning

confidence: 99%

“…Conjugation was performed as described in Felczak et al [ 47 ] except that the E. coli strain WM6026 was used as a donor of pRL814. Briefly, Z. mobilis strains were inoculated from a single colony and grown in 5 ml of ZRMG media statically in 14-ml tubes at 30 °C.…”

Section: Methodsmentioning

confidence: 99%

Zymomonas diversity and potential for biofuel production

Felczak

Bowers

Woyke

et al. 2021

Biotechnol Biofuels

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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.

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“…Zymomonas mobilis is a facultative anaerobic Gram-negative microorganism, well known for its efficient production of bioethanol as replacement for fossil fuels ( Swings and De Ley, 1977 ; Skotnicki et al, 1982 ; Sprenger, 1996 ; Gunasekaran and Raj, 1999 ; He et al, 2014 ; Yang et al, 2016a ). The bacterium possesses a relatively simple central metabolic network, including a non-functional Embden-Meyerhof-Parnas (EMP) glycolytic route ( Fuhrer et al, 2005 ; Seo et al, 2005 ; Felczak et al, 2019 ), incomplete pentose phosphate pathway ( Feldmann et al, 1992 ; De Graaf et al, 1999 ) and truncated tricarboxylic acid (TCA) cycle ( Lee et al, 2010 ; Jacobson et al, 2019 ). Instead, hexoses including glucose and fructose are metabolized via the Entner-Doudoroff (ED) pathway to form primarily ethanol, along with glycerol and succinic, lactic and acetic acid by-products ( Zhang et al, 2019b ).…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic and Kinetic Modeling of Co-utilization of Glucose and Xylose for 2,3-BDO Production by Zymomonas mobilis

Spiller

Dowe

et al. 2021

Front. Bioeng. Biotechnol.

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Prior engineering of the ethanologen Zymomonas mobilis has enabled it to metabolize xylose and to produce 2,3-butanediol (2,3-BDO) as a dominant fermentation product. When co-fermenting with xylose, glucose is preferentially utilized, even though xylose metabolism generates ATP more efficiently during 2,3-BDO production on a BDO-mol basis. To gain a deeper understanding of Z. mobilis metabolism, we first estimated the kinetic parameters of the glucose facilitator protein of Z. mobilis by fitting a kinetic uptake model, which shows that the maximum transport capacity of glucose is seven times higher than that of xylose, and glucose is six times more affinitive to the transporter than xylose. With these estimated kinetic parameters, we further compared the thermodynamic driving force and enzyme protein cost of glucose and xylose metabolism. It is found that, although 20% more ATP can be yielded stoichiometrically during xylose utilization, glucose metabolism is thermodynamically more favorable with 6% greater cumulative Gibbs free energy change, more economical with 37% less enzyme cost required at the initial stage and sustains the advantage of the thermodynamic driving force and protein cost through the fermentation process until glucose is exhausted. Glucose-6-phosphate dehydrogenase (g6pdh), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and phosphoglycerate mutase (pgm) are identified as thermodynamic bottlenecks in glucose utilization pathway, as well as two more enzymes of xylose isomerase and ribulose-5-phosphate epimerase in xylose metabolism. Acetolactate synthase is found as potential engineering target for optimized protein cost supporting unit metabolic flux. Pathway analysis was then extended to the core stoichiometric matrix of Z. mobilis metabolism. Growth was simulated by dynamic flux balance analysis and the model was validated showing good agreement with experimental data. Dynamic FBA simulations suggest that a high agitation is preferable to increase 2,3-BDO productivity while a moderate agitation will benefit the 2,3-BDO titer. Taken together, this work provides thermodynamic and kinetic insights of Z. mobilis metabolism on dual substrates, and guidance of bioengineering efforts to increase hydrocarbon fuel production.

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Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4

Cited by 13 publications

References 22 publications

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

Zymomonas diversity and potential for biofuel production

Thermodynamic and Kinetic Modeling of Co-utilization of Glucose and Xylose for 2,3-BDO Production by Zymomonas mobilis

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