Fermentation of dihydroxyacetone by engineered
            <i>Escherichia coli</i>
            and
            <i>Klebsiella variicola</i>
            to products

Wang, Liang; Chauliac, Diane; Rhee, Mun Su; Panneerselvam, A.; Ingram, L. O.; Shanmugam, K. T.

doi:10.1073/pnas.1801002115

Cited by 9 publications

(8 citation statements)

References 36 publications

(41 reference statements)

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“…This has indeed been observed recently in an E. coli strain that overexpresses an ATP-dependent DHA kinase during anaerobic growth on DHA (47). However, this was not observed under our aerobic conditions, i.e., overexpressing glpK (i.e., glpK +++) decreased the growth rate (Fig.…”

Section: Resultssupporting

confidence: 71%

“…Enabling the rational design of strains that use DHA efficiently should improve biotechnological applications involving DHA as an intermediate, a well-known example being the bioconversion of glycerol into value-added chemicals (56). This should also facilitate research aiming at constructing synthetic methylotrophs (i.e., engineering of nonnative methylotrophs for methane and methanol-based production of chemicals) for which DHA can be an intermediate (47, 57).…”

Section: Resultsmentioning

confidence: 99%

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Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli

Peiro

Millard

Simone

et al. 2019

Appl Environ Microbiol

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In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous, and important intermediate for various chemicals in industry, by analyzing its metabolism at the system level in Escherichia coli. Using constraint-based modeling, we show that the growth of E. coli on DHA is suboptimal and identify the potential causes. Nuclear magnetic resonance analysis shows that DHA is degraded nonenzymatically into substrates known to be unfavorable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e., the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobically mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA. IMPORTANCE DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli. We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals.

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Section: Resultssupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli

Peiro

Millard

Simone

et al. 2019

Appl Environ Microbiol

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“…A similar effect of FBP on Z. mobilis pyruvate kinase or inactivation of another enzyme cannot be excluded. Toxicity of DHAP and DHA to the cells may also be a reason for the observed decrease in growth rate of tpi -overexpressing strains (Bar-Even et al, 2012; Wang et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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Zymomonas mobilis is a bacterium that produces ethanol from glucose at up to 97% of theoretical efficiency on a carbon basis. One factor contributing to the high efficiency of ethanol production is that Z. mobilis has a low biomass yield. The low biomass yield may be caused partly by the low ATP yield of the Entner-Doudoroff (ED) glycolytic pathway used by Z. mobilis, which produces only one ATP per glucose consumed. To test the hypothesis that ATP yield limits biomass yield in Z. mobilis, we attempted to introduce the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (with double the ATP yield) by expressing phosphofructokinase (Pfk I) from Escherichia coli. Expression of Pfk I caused growth inhibition and resulted in accumulation of mutations in the pfkA gene. Co-expression of additional EMP enzymes, fructose bisphosphate aldolase (Fba) and triose phosphate isomerase (Tpi), with Pfk I did not enable EMP flux, and resulted in production of glycerol as a side product. Further analysis indicated that heterologous reactions may have operated in the reverse direction because of native metabolite concentrations. This study reveals how the metabolomic context of a chassis organism influences the range of pathways that can be added by heterologous expression.

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“…d ‐LDH from Lactobacillus plantarum is completely inactivated after incubation at 50 °C . Studies of d ‐LDHs from Bacillus coagulans support this observation . Therefore, the limited activity of these d ‐LDHs has precluded their use in high‐temperature d ‐lactic acid production.…”

Section: Figurementioning

confidence: 91%

Carbon Flux Trapping: Highly Efficient Production of Polymer‐Grade d‐Lactic Acid with a Thermophilic d‐Lactate Dehydrogenase

Tao

2016

ChemBioChem

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High production of polymer-grade d-lactic acid is urgently required, particularly for the synthesis of polylactic acid. High-temperature fermentation has multiple advantages, such as lower equipment requirement and energy consumption, which are essential for lowering operating costs. We identified and introduced a unique d-lactate dehydrogenase into a thermotolerant butane-2,3-diol-producing strain. Carbon flux "trapping" was achieved by a "trapping point" created by combination of the introduced enzyme and the host efflux pump, which afforded irreversible transport of d-lactic acid. The overall carbon flux of the engineered strain was significantly enhanced and was redistributed predominantly to d-lactic acid. Under optimized conditions at 50 °C, d-lactic acid reached the highest titer (226.6 g L(-1) ) reported to date. This discovery allows us to extend the carbon flux trapping strategy to engineering complex metabolic networks.

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Fermentation of dihydroxyacetone by engineered Escherichia coli and Klebsiella variicola to products

Cited by 9 publications

References 36 publications

Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli

Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli

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

Carbon Flux Trapping: Highly Efficient Production of Polymer‐Grade d‐Lactic Acid with a Thermophilic d‐Lactate Dehydrogenase

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