Characterization of Heterologous and Native Enzyme Activity Profiles in Metabolically Engineered Zymomonas Mobilis Strains During Batch Fermentation of Glucose and Xylose Mixtures

Gao, Qiang; Zhang, Min; McMillan, James D.; Kompala, Dhinakar S.

doi:10.1007/978-1-4612-0119-9_28

Cited by 6 publications

(6 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Xylose isomerase (XI) activity was assayed in a reaction mixture containing 1 mM triethanolamine (TEA) (pH 7.5), 0.3 mM NADH, 100 mM xylose, 10 mM MgSO 4 , 4 U/mL SDH (Roche Diagnostics, Indianapolis, IN) and 10 mL of crude cell extract (Gao et al, 2002;Schellenberg et al, 1984). Xylulokinase was assayed by a method a described in Akinterinwa and Cirino (2009).…”

Section: Enzymatic Assaysmentioning

confidence: 99%

“…An important breakthrough came when Zhang et al (1995) successfully applied metabolic engineering to expand its substrate range to xylose. In the ensuing decade, however, xylose metabolism continued to be a focal point of research as it was found that the ability of engineered strains to metabolize xylose was considerably lower than that of glucose and improvement was needed (De Graaf et al, 1999;Gao et al, 2002;Jeon et al, 2005;Kim et al, 2000).…”

Section: Introductionmentioning

confidence: 97%

See 1 more Smart Citation

Adaptation yields a highly efficient xylose‐fermenting Zymomonas mobilis strain

Agrawal

Mao

Chen

2010

Biotech & Bioengineering

View full text Add to dashboard Cite

Zymomonas mobilis is a superb ethanol producer with productivity exceeding yeast strains by several fold. Although metabolic engineering was successfully applied to expand its substrate range to include xylose, xylose fermentation lagged far behind glucose. In addition, xylose fermentation was often incomplete when its initial concentration was higher than 5%. Improvement of xylose fermentation is therefore necessary. In this work, we applied adaptation to improve xylose fermentation in metabolically engineered strains. As a result of adaptation over 80 days and 30 serial transfers in a medium containing high concentration of xylose, a strain, referred as A3, with markedly improved xylose metabolism was obtained. The strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, a total of 9% (w/v) ethanol was obtained from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Further investigation reveals evidence for an altered xylitol metabolism in A3 with reduced xylitol formation. Additionally xylitol tolerance in A3 was increased. Furthermore, xylose isomerase activity was increased by several times in A3, allowing cells to channel more xylose to ethanol than to xylitol. Taken together, these results strongly suggest that altered xylitol metabolism is key to improved xylose metabolism in adapted A3 strain. This work further demonstrates that adaptation and metabolic engineering can be used synergistically for strain improvement.

show abstract

Section: Enzymatic Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Adaptation yields a highly efficient xylose‐fermenting Zymomonas mobilis strain

Agrawal

Mao

Chen

2010

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Total cellular protein was assumed to be 67% of dry weight for growing Z. mobilis cells (Low and Rogers, 1984;Swings and De Ley, 1977;Gao et al, 2002).…”

Section: Dynamic Balance Equationsmentioning

confidence: 99%

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis

Altintas

Eddy

Zhang

et al. 2006

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.

show abstract

“…Xylose isomerase activity was determined based on NADH oxidation by sorbitol dehydrogenase as previously described (14). Xylulokinase assays were performed as reported elsewhere (12).…”

Section: Dna Manipulationsmentioning

confidence: 99%

Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

Kawaguchi

Vertès

Okino

et al. 2006

Appl Environ Microbiol

216

140

View full text Add to dashboard Cite

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7: [182][183][184][185][186][187][188][189][190][191][192][193][194][195][196] 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

show abstract

Characterization of Heterologous and Native Enzyme Activity Profiles in Metabolically Engineered Zymomonas Mobilis Strains During Batch Fermentation of Glucose and Xylose Mixtures

Cited by 6 publications

References 10 publications

Adaptation yields a highly efficient xylose‐fermenting Zymomonas mobilis strain

Adaptation yields a highly efficient xylose‐fermenting Zymomonas mobilis strain

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis

Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

Contact Info

Product

Resources

About