9 research outputs found
Optimization of ethylene glycol production from (d)-xylose via a synthetic pathway implemented in Escherichia coli
BACKGROUND: Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable. RESULTS: Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of (d)-xylose isomerase, (d)-xylulose-1-kinase, (d)-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD. The production strain was optimized by deleting the genes encoding for (d)-xylulose-5 kinase (xylB) and glycolaldehyde dehydrogenase (aldA), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20Â g/L EG with a molar yield and productivity of 0.91 and 0.37Â g/(L.h), respectively, in a controlled bioreactor under aerobic conditions. CONCLUSIONS: We have demonstrated the feasibility to produce EG from (d)-xylose via a synthetic pathway in E. coli at approximately 90Â % of the theoretical yield. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0312-7) contains supplementary material, which is available to authorized users
Optimization of ethylene glycol production from (d)-xylose via a synthetic pathway implemented in Escherichia coli
The synthetic xylulose-1 phosphate pathway increases production of glycolic acid from xylose-rich sugar mixtures
Identificação de resĂduos de aminoĂĄcidos envolvidos no transporte ativo de açĂșcares pela permease AGT1 de saccharomyces cerevisiae
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de CiĂȘncias BiolĂłgicas. Programa de PĂłs-graduação em BiotecnologiaEm Saccharomyces cerevisiae, a permease codificada pelo gene AGT1 Ă© responsĂĄvel pelo transporte ativo de uma sĂ©rie de a-glicosĂdeos usados em diversas aplicaçÔes industriais de leveduras, como panificação, cervejaria, produção de bebidas destiladas e ĂĄlcool combustĂvel. O transporte destes açĂșcares para dentro da cĂ©lula, atravĂ©s de proteĂnas transportadoras, Ă© o passo inicial para seu metabolismo, e tambĂ©m constitui um fator limitante na fermentação do açĂșcar. Com o intuito de compreender detalhes moleculares do mecanismo de transporte e contribuir para a otimização do processo fermentativo, no presente trabalho foi analisado os resĂduos de aminoĂĄcidos da permease Agt1p que poderiam estar envolvidos na ligação do prĂłton e/ou especificidade do substrato. Analisando a estrutura prevista para o transportador Agt1p, identificamos 4 resĂduos de aminoĂĄcidos carregados em seus segmentos transmembrana (Glu-120, Asp-123, Glu-167 e Arg-504), resĂduos que estĂŁo significativamente conservados nas mesmas posiçÔes em outros genes de transportadores de a-glicosĂdeos em Saccharomyces, Torulaspora, Kluyveromyces e Candida. Permeases Agt1p mutantes nos resĂduos Glu-120, Asp-123 e Arg-504 foram geradas por mutagĂȘnese sĂtio-dirigida, expressadas em uma cepa agt1?, e a funcionalidade das permeases foi testada atravĂ©s do crescimento em maltotriose. A substituição do resĂduo Arg-504 pela alanina aboliu completamente a utilização de maltotriose pelas cĂ©lulas, enquanto que as permeases mutantes geradas pela substituição do Asp-123 pela glicina ou do Glu-120 pela alanina ocasionaram menores taxas de consumo de maltotriose e menor rendimento de etanol quando comparado ao transportador Agt1p normal, porĂ©m nĂŁo impediram o crescimento em maltotriose. A atividade de transporte da permease Agt1p normal ou dos mutantes derivados tambĂ©m foi avaliada pelo transporte de pNPaG (substrato especĂfico para o transportador Agt1p) e pelo transporte ativo de outros a-glicosĂdeos (maltose, maltotriose, trealose, sacarose e a-metil glicosĂdeo). Nesses ensaios, a mutação R504A ocasionou a maior perda na atividade de transporte da permease, tendo em vista que apresentou taxas similares Ă s cĂ©lulas sem a permease Agt1p. Em contrapartida, os mutantes E120A e D123G apresentaram atividades de transporte de pNPaG e de co-transporte açĂșcar-H+ inferiores Ă observada para o transportador Agt1p normal, condizendo com o perfil de utilização de maltotriose. Apesar das diferentes atividades de transporte encontradas para as permeases construĂdas, todas essas permeases (normal ou mutantes) quando fusionadas Ă GFP em sua extremidade C-terminal apresentaram-se normalmente localizadas na membrana plasmĂĄtica. Portanto, os resultados demonstram o envolvimento dos resĂduos mutados no transporte ativo de açĂșcares realizado pela permease Agt1p, sugerindo a participação dos resĂduos Glu-120 e Asp-123 na translocação do prĂłton, e de Arg-504 na ligação do substrato. PorĂ©m, estudos ainda sĂŁo necessĂĄrios para melhor esclarecer o mecanismo de transporte ativo realizado pela permease Agt1p, o que certamente Ă© de grande relevĂąncia para o desenvolvimento de estratĂ©gias que permitam a otimização dos processos fermentativos industriais
Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permeaseâż
Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known α-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (Km, 36 ± 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations
Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)âXylose into Value-Added Chemicals
A synthetic pathway for (d)-xylose assimilation was stoichiometrically
evaluated and implemented in Escherichia coli strains. The pathway proceeds via isomerization of (d)-xylose
to (d)-xylulose, phosphorylation of (d)-xylulose
to obtain (d)-xylulose-1-phosphate (X1P), and aldolytic cleavage
of the latter to yield glycolaldehyde and DHAP. Stoichiometric analyses
showed that this pathway provides access to ethylene glycol with a
theoretical molar yield of 1. Alternatively, both glycolaldehyde and
DHAP can be converted to glycolic acid with a theoretical yield that
is 20% higher than for the exclusive production of this acid via the
glyoxylate shunt. Simultaneous expression of xylulose-1 kinase and
X1P aldolase activities, provided by human ketohexokinase-C and human
aldolase-B, respectively, restored growth of a (d)-xylulose-5-kinase
mutant on xylose. This strain produced ethylene glycol as the major
metabolic endproduct. Metabolic engineering provided strains that
assimilated the entire C2 fraction into the central metabolism or
that produced 4.3 g/L glycolic acid at a molar yield of 0.9 in shake
flasks
Additional file 1. of Optimization of ethylene glycol production from (d)-xylose via a synthetic pathway implemented in Escherichia coli
Log2 transformed expression levels of candidate glycolaldehyde reductase