Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for d-mannitol formation in a whole-cell biotransformation

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 m, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)؊1 day ؊1 with a 90% molar yield over a 45-h production period.Microbes in nature are commonly in surface-associated, matrix-enclosed structures referred to as biofilms (7,28). Bacteria in biofilms can differ profoundly in terms of phenotypic characteristics (31, 33) or adaptive responses to stress (17, 38) from their planktonic counterparts, which can provide survival advantages and protection in a range of environmental conditions (16,23). Biofilms are also commonly found on medical implants and are responsible for many persistent infections due to their increased resistance to antimicrobial agents (13).While deleterious in clinical settings, biofilms are of great interest in biotechnology. For example, biofilms have been used as biocatalysts in bioremediation and wastewater treatment processes (21,23,27) and in fermentative vinegar (9) and ethanol (20) production from agricultural materials, as well as in biosynthesis of polymers (42). To our knowledge, the use of biofilms for transformation of fine chemicals into value-added products has not been reported previously.Biotransformation using viable cells capable of cofactor regeneration is an important approach to fine-chemical production (30, 34). A common obstacle in biocatalytic fine-chemical production is the potential toxic effects of substrates and/or products on cells during the process. S...

Section: Figmentioning

confidence: 99%

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Li¹,

Webb

Kjelleberg

et al. 2006

“…To further increase the intracellular NADH driving force, formate dehydrogenase (Fdh) from C. boidinii (1) carried on plasmid pCS138 was overexpressed to oxidize formate into CO 2 and NADH (8,28,32). The production profiles of the strains (JCL166 transformed with plasmids pEL11 and pIM8) with and without C. boidinii Fdh overexpression are compared in Fig.…”

Section: Resultsmentioning

confidence: 99%

Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

Shen

Lan

Dekishima

et al. 2011

565

508

1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

“…GLF not only has a high affinity for glucose but also uses mannose as a substrate (29,47). GLF was repeatedly heterologously expressed in E. coli to complement mutants defective in glucosespecific PTS components (22,29,47) and, since GLF is primarily a glucose transporter, it was assumed that it will also confer glucose utilization to R. eutropha.…”

Section: Resultsmentioning

confidence: 99%

Extension of the Substrate Utilization Range of Ralstonia eutropha Strain H16 by Metabolic Engineering To Include Mannose and Glucose

Sichwart

Hetzler

Bröker

et al. 2011

The Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha strain H16 is known for its narrow carbohydrate utilization range, which limits its use for biotechnological production of polyhydroxyalkanoates and possibly other products from renewable resources. To broaden its substrate utilization range, which is for carbohydrates and related compounds limited to fructose, N-acetylglucosamine, and gluconate, strain H16 was engineered to use mannose and glucose as sole carbon sources for growth. The genes for a facilitated diffusion protein (glf) from Zymomonas mobilis and for a glucokinase (glk), mannofructokinase (mak), and phosphomannose isomerase (pmi) from Escherichia coli were alone or in combination constitutively expressed in R. eutropha strain H16 under the control of the neokanamycin or lac promoter, respectively, using an episomal broad-host-range vector. Recombinant strains harboring pBBR1MCS-3::glf::mak::pmi or pBBR1MCS-3::glf::pmi grew on mannose, whereas pBBR1MCS-3::glf::mak and pBBR1MCS-3::glf did not confer the ability to utilize mannose as a carbon source to R. eutropha. The recombinant strain harboring pBBR1MCS-3::glf::pmi exhibited slower growth on mannose than the recombinant strain harboring pBBR1MCS-3::glf::mak::pmi. These data indicated that phosphomannose isomerase is required to convert mannose-6-phosphate into fructose-6-phosphate for subsequent catabolism via the Entner-Doudoroff pathway. In addition, all plasmids also conferred to R. eutropha the ability to grow in the presence of glucose. The best growth was observed with a recombinant R. eutropha strain harboring plasmid pBBR1MCS-2::P nk ::glk::glf. In addition, expression of the respective enzymes was demonstrated at the transcriptional and protein levels and by measuring the activities of mannofructokinase (0.622 ؎ 0.063 U mg ؊1 ), phosphomannose isomerase (0.251 ؎ 0.017 U mg ؊1 ), and glucokinase (0.518 ؎ 0.040 U mg ؊1 ). Cells of recombinant strains of R. eutropha synthesized poly(3-hydroxybutyrate) to ca. 65 to 67% (wt/wt) of the cell dry mass in the presence of 1% (wt/vol) glucose or mannose as the sole carbon sources.The facultative chemolithoautotrophic Gram-negative Ralstonia eutropha strain H16, formerly known as Alcaligenes eutrophus, serves as model organism for studying the metabolism of poly(3-hydroxybuyrate) (PHB) and hydrogen-based chemolithoautotrophy. If grown autotrophically, strain H16 assimilates CO 2 via the Calvin-Benson-Bassham cycle. It uses also various organic compounds as a carbon and energy source if H 2 is absent. Under unbalanced growth conditions, PHB is accumulated in granules as a storage compound for carbon and energy (8). This is the case when a carbon source is abundant and if another macroelement such as nitrogen is lacking. The well-understood physiology of this "Knallgas" bacterium, its ability to be cultivated to high cell densities also at large scale, and the enormous polyhydroxyalkanoate (PHA) content of the cells led the industry consider to producing PHAs by fermentatio...