Abstract:Escherichia coli Δglk ΔmanZ ΔptsG glucose strains that lack the glucose phosphotransferase system (PTS) and the mannose PTS as well as glucokinase have been widely used by researchers studying the PTS. In this study we show that both fast- and slow-growing spontaneous glucose revertants can be readily obtained from Δglk ΔmanZ ΔptsG glucose strains. All of the fast-growing revertants either altered the N-acetylglucosamine PTS or caused its overproduction by inactivating the NagC repressor protein, which regulat… Show more
“…The cell density increased or decreased in proportion to the total quantity of metabolizable carbon/energy sources. During the process, the concentration of the three sugars remained at 10 mM, demonstrating that they were not consumed over the course of the 4 days, and that glucose-degrading mutants did not arise spontaneously [5]. Since the strains never encountered high and potentially toxic concentrations of the aromatic substrates, such a continuous process using substrate-selective microbes would be a very effective means to remove a portion of the carbon sources from a variable feed stream.Fig.…”
Background
Lignocellulosic biomass is an attractive, inexpensive source of potentially fermentable sugars. However, hydrolysis of lignocellulose results in a complex mixture containing microbial inhibitors at variable composition. A single microbial species is unable to detoxify or even tolerate these non-sugar components while converting the sugar mixtures effectively to a product of interest. Often multiple substrates are metabolized sequentially because of microbial regulatory mechanisms. To overcome these problems, we engineered strains of
Acinetobacter baylyi
ADP1 to comprise a consortium able to degrade benzoate and 4-hydroxybenzoate simultaneously under batch and continuous conditions in the presence of sugars. We furthermore used a thermotolerant yeast,
Kluyveromyces marxianus
, to convert the glucose remaining after detoxification to ethanol.
Results
The two engineered strains, one unable to metabolize benzoate and another unable to metabolize 4-hydroxybenzoate, when grown together removed these two inhibitors simultaneously under batch conditions. Under continuous conditions, a single strain with a deletion in the
gcd
gene metabolized both inhibitors in the presence of sugars. After this batch detoxification using ADP1-derived mutants,
K. marxianus
generated 36.6 g/L ethanol.
Conclusions
We demonstrated approaches for the simultaneous removal of two aromatic inhibitors from a simulated lignocellulosic hydrolysate. A two-stage batch process converted the residual sugar into a non-growth-associated product, ethanol. Such a two-stage process with bacteria (
A. baylyi)
and yeast (
K. marxianus
) is advantageous, because the yeast fermentation occurs at a higher temperature which prevents growth and ethanol consumption of
A. baylyi.
Conceptually, the process can be extended to other inhibitors or sugars found in real hydrolysates. That is, additional strains which degrade components of lignocellulosic hydrolysates could be made substrate-selective and targeted for use with specific complex mixtures found in a hydrolysate.
Electronic supplementary material
The online version of this article (10.1186/s13068-019-1434-7) contains supplementary material, which is available to authorized users.
“…The cell density increased or decreased in proportion to the total quantity of metabolizable carbon/energy sources. During the process, the concentration of the three sugars remained at 10 mM, demonstrating that they were not consumed over the course of the 4 days, and that glucose-degrading mutants did not arise spontaneously [5]. Since the strains never encountered high and potentially toxic concentrations of the aromatic substrates, such a continuous process using substrate-selective microbes would be a very effective means to remove a portion of the carbon sources from a variable feed stream.Fig.…”
Background
Lignocellulosic biomass is an attractive, inexpensive source of potentially fermentable sugars. However, hydrolysis of lignocellulose results in a complex mixture containing microbial inhibitors at variable composition. A single microbial species is unable to detoxify or even tolerate these non-sugar components while converting the sugar mixtures effectively to a product of interest. Often multiple substrates are metabolized sequentially because of microbial regulatory mechanisms. To overcome these problems, we engineered strains of
Acinetobacter baylyi
ADP1 to comprise a consortium able to degrade benzoate and 4-hydroxybenzoate simultaneously under batch and continuous conditions in the presence of sugars. We furthermore used a thermotolerant yeast,
Kluyveromyces marxianus
, to convert the glucose remaining after detoxification to ethanol.
Results
The two engineered strains, one unable to metabolize benzoate and another unable to metabolize 4-hydroxybenzoate, when grown together removed these two inhibitors simultaneously under batch conditions. Under continuous conditions, a single strain with a deletion in the
gcd
gene metabolized both inhibitors in the presence of sugars. After this batch detoxification using ADP1-derived mutants,
K. marxianus
generated 36.6 g/L ethanol.
Conclusions
We demonstrated approaches for the simultaneous removal of two aromatic inhibitors from a simulated lignocellulosic hydrolysate. A two-stage batch process converted the residual sugar into a non-growth-associated product, ethanol. Such a two-stage process with bacteria (
A. baylyi)
and yeast (
K. marxianus
) is advantageous, because the yeast fermentation occurs at a higher temperature which prevents growth and ethanol consumption of
A. baylyi.
Conceptually, the process can be extended to other inhibitors or sugars found in real hydrolysates. That is, additional strains which degrade components of lignocellulosic hydrolysates could be made substrate-selective and targeted for use with specific complex mixtures found in a hydrolysate.
Electronic supplementary material
The online version of this article (10.1186/s13068-019-1434-7) contains supplementary material, which is available to authorized users.
“…The glucose transport system in E. coli is known as follows. There are glucose PTS [ 19 ], mannose PTS [ 20 ], maltose PTS [ 21 ], and N -acetylglucosamine PTS [ 2 ]. Non-PTS transporters include Mal ABC transporter [ 3 ], ExuT [ 13 ], Mgl ABC transporter [ 22 ], and GalP [ 9 , 23 ] ( Fig.…”
Section: Discussionmentioning
confidence: 99%
“…Several studies have been performed to discover an alternative route for transporting glucose by growing a strain that cannot use glucose under various conditions. Crigler et al [2] analyzed the characteristics of glk-, manZ-, and ptsG-deleted E. coli cells on a minimal medium containing glucose and reported that glucose can be transported through N-acetylglucosamine PTS.…”
Section: Introductionmentioning
confidence: 99%
“…Sugars can be actively transported into microbial cells through various transport systems, including phosphotransferase system (PTS), ATP-binding cassette (ABC) transporter, and cation gradient-driven symporter [ 1 , 2 ]. PTS, one of the most efficient sugar transport systems, consists of non-sugar-specific common enzymes, namely, enzyme I (EI), phosphohistidine carrier protein (HPr), and sugar-specific enzyme II complexes [ 3 ].…”
Section: Introductionmentioning
confidence: 99%
“…Crigler et al . [ 2 ] analyzed the characteristics of glk -, manZ -, and ptsG -deleted E. coli cells on a minimal medium containing glucose and reported that glucose can be transported through N -acetylglucosamine PTS.…”
When ptsG, a glucose-specific phosphotransferase system (PTS) component, is deleted in Escherichia coli, growth can be severely poor because of the lack of efficient glucose transport. We discovered a new PTS transport system that could transport glucose through the growth-coupled experimental evolution of ptsG-deficient E. coli C strain under anaerobic conditions. Genome sequencing revealed mutations in agaR, which encodes a repressor of N-acetylgalactosamine (Aga) PTS expression in evolved progeny strains. RT-qPCR analysis showed that the expression of Aga PTS gene increased because of the loss-of-function of agaR. We confirmed the efficient Aga PTS-mediated glucose uptake by genetic complementation and anaerobic fermentation. We discussed the discovery of new glucose transporter in terms of different genetic backgrounds of E. coli strains, and the relationship between the pattern of mixed-acids fermentation and glucose transport rate.
L-Methionine is the unique sulfur-containing amino acid essential for humans and animals, which is widely used in pharmaceutical, food and feed industries. Its green and efficient production has attracted a great deal of attention. Fermentation-enzymatic coupling route is regarded to be promising for L-methionine biosynthesis, while O-succinyl-L-homoserine mercaptotransferase (MetZ) is the key biocatalyst. Exploring and engineering of MetZ with ideal catalytic properties is highly desired. Herein, the MetZ from Chromobacterium violaceum (CvMetZ) was screened and through in silico analyses, potential beneficial amino acid residues both at the access channel and around the oxygen anion holes were anchored for site-saturation mutagenesis. The positive mutants were obtained with improved activity for L-methionine biosynthesis using O-succinyl-Lhomoserine (OSH) and methyl mercaptan as substrate. The best mutant was further applied for L-methionine production and supply of methyl mercaptan was optimized in a fed-batch approach. The conversion of 100 g/L OSH reached 92% with L-methionine yield of 62.6 g/L, which was well coupled with the OSH fermentation level.
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