Butanol production from glycerol by recombinant Escherichia coli

Escherichia coli MG1655 (DE3) with the ability to synthesize butanol from glycerol was constructed by metabolic engineering. The genes thil, adhe2, bcs operon (crt, bcd, etfB, etfA, and hbd) were cloned into the plasmid vectors, pETDuet-1 and pACYCDuet-1, then the two resulting plasmids, pACYC-thl-bcs and pET-adhe2, were transferred to E. coli, and the recombinant strain was able to synthesize up to 18.5 mg/L butanol on a glycerol-containing medium. After the glycerol transport protein gene GlpF was expressed, the butanol production was improved to 22.7 mg/L. The competing pathway of byproducts, such as ethanol, succinate, and lactate, was subsequently deleted to improve the 1-butanol production to 97.9 mg/L. Moreover, a NADH regeneration system was introduced into the E. coli, and finally a 154.0 mg/L butanol titer was achieved in a laboratory-scale shake-flask experiment.     

Metabolic engineering of Propionibacterium freudenreichii for n-propanol production

Propionibacteria are widely used in industry for manufacturing of Swiss cheese, vitamin B₁₂, and propionic acid. However, little is known about their genetics and only a few reports are available on the metabolic engineering of propionibacteria aiming at enhancing fermentative production of vitamin B₁₂ and propionic acid. n-Propanol is a common solvent, an intermediate in many industrial applications, and a promising biofuel. To date, no wild-type microorganism is known to produce n-propanol in sufficient quantities for industrial application purposes. In this study, a bifunctional aldehyde/alcohol dehydrogenase (adhE) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. The mutants expressing the adhE gene converted propionyl- coenzyme A, which is the precursor for propionic acid biosynthesis, to n-propanol. The production of n-propanol was limited by NADH availability, which was improved significantly by using glycerol as the carbon source. Interestingly, the improved propanol production was accompanied by a significant increase in propionic acid productivity, indicating a positive effect of n-propanol biosynthesis on propionic acid fermentative production. To our best knowledge, this is the first report on producing n-propanol by metabolically engineered propionibacteria, which offers a novel route to produce n-propanol from renewable feedstock, and possibly a new way to boost propionic acid fermentation.     

Improvement in ethanol productivity of engineered <Emphasis Type="Italic">E. coli</Emphasis> strain SSY13 in defined medium via adaptive evolution

E. coli has the ability to ferment both C5 and C6 sugars and produce mixture of acids along with small amount of ethanol. In our previous study, we reported the construction of an ethanologenic E. coli strain by modulating flux through the endogenous pathways. In the current study, we made further changes in the strain to make the overall process industry friendly; the changes being (1) removal of plasmid, (2) use of low-cost defined medium, and (3) improvement in consumption rate of both C5 and C6 sugars. We first constructed a plasmid-free strain SSY13 and passaged it on AM1–xylose minimal medium plate for 150 days. Further passaging was done for 56 days in liquid AM1 medium containing either glucose or xylose on alternate days. We observed an increase in specific growth rate and carbon utilization rate with increase in passage numbers until 42 days for both glucose and xylose. The 42nd day passaged strain SSK42 fermented 113 g/L xylose in AM1 minimal medium and produced 51.1 g/L ethanol in 72 h at 89% of maximum theoretical yield with ethanol productivity of 1.4 g/L/h during 24–48 h of fermentation. The ethanol titer, yield and productivity were 49, 40 and 36% higher, respectively, for SSK42 as compared to unevolved SSY13 strain.     

Metabolic engineering of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and in silico investigation for enhanced 2,3-butanediol production

Objectives

To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated.

Results

Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta-ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation.

Conclusions

K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity.

     

Extracellular recombinant protein production under continuous culture conditions with Escherichia coli using an alternative plasmid selection mechanism

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process. To avoid the use of antibiotics and render this process more sustainable, alternative plasmid selection mechanisms were required. In the current study, the strain E. coli JM109 harboring plasmid p582 was shown to stably express and secrete recombinant β-glucanase in continuous culture using a minimal medium. The segregational instability of the plasmid in the absence of antibiotic selection pressure was demonstrated. The leuB gene, crucial in the leucine biosynthetic pathway, was cloned onto plasmid p582 and the new construct transformed into an E. coli Keio (ΔleuB) knockout strain. The ability of the construct to complement the leucine auxotrophy was initially tested in shake-flasks and batch cultivation. Later, this strain was successfully grown for more than 200 h in a chemostat and was found to be able to express the recombinant protein. Significantly, it showed a stable maintenance of the recombinant plasmid in the absence of any antibiotics. The plasmid stability in a continuously cultivated E. coli fermentation, in the absence of antibiotics, with extracellular secretion of recombinant protein provides an interesting model for further improvements.     

Draft Genome Sequence of the Flocculating Zymomonas mobilis Strain ZM401 (ATCC 31822)

Zymomonas mobilis ZM401 is a flocculating strain which can be self-immobilized within fermentors for a high-cell-density culture to improve ethanol productivity, as well as high-gravity fermentation to increase ethanol titer, due to its improved ethanol tolerance associated with the morphological change. Here, we report its draft genome sequence.     

Fructophilic Characteristics of Fructobacillus spp. may be due to the Absence of an Alcohol/Acetaldehyde Dehydrogenase Gene (adhE)

Fructophilic strains of Leuconostoc spp. have recently been reclassified to a new genus, i.e., Fructobacillus. Members of the genus are differentiated from Leuconostoc spp. by their preference for fructose on growth, requirement of an electron acceptor for glucose metabolism, and the inability to produce ethanol from the fermentation of glucose. In the present study, enzyme activities and genes involved in ethanol production were studied, since this is the key pathway for NAD⁺/NADH cycling in heterofermentative lactic acid bacteria. Fructobacillus spp. has a weak alcohol dehydrogenase activity and has no acetaldehyde dehydrogenase activity, whereas both enzymes are active in Leuconostoc mesenteroides. The bifunctional alcohol/acetaldehyde dehydrogenase gene, adhE, was described in Leuconostoc spp., but not in Fructobacillus spp. These results suggested that, due to the deficiency of the adhE gene, the normal pathway for ethanol production is absent in Fructobacillus spp. This leads to a shortage of NAD⁺, and the requirement for an electron acceptor in glucose metabolism. Fructophilic characteristics, as observed for Fructobacillus spp., are thus due to the absence of the adhE gene, and a phenotype that most likely evolved as a result of regressive evolution.     

Production of 3-hydroxypropionic acid from glycerol by acid tolerant Escherichia coli

The biological production of 3-hydroxypropionic acid (3-HP) has attracted significant attention because of its industrial importance. The low titer, yield and productivity, all of which are related directly or indirectly to the toxicity of 3-HP, have limited the commercial production of 3-HP. The aim of this study was to identify and select a 3-HP tolerant Escherichia coli strain among nine strains reported to produce various organic acids efficiently at high titer. When transformed with heterologous glycerol dehydratase, reactivase and aldehyde dehydrogenase, all nine E. coli strains produced 3-HP from glycerol but the level of 3-HP production, protein expression and activities of the important enzymes differed significantly according to the strain. Two E. coli strains, W3110 and W, showed higher levels of growth than the others in the presence of 25 g/L 3-HP. In the glycerol fed-batch bioreactor experiments, the recombinant E. coli W produced a high level of 3-HP at 460 ± 10 mM (41.5 ± 1.1 g/L) in 48 h with a yield of 31 % and a productivity of 0.86 ± 0.05 g/L h. In contrast, the recombinant E. coli W3110 produced only 180 ± 8.5 mM 3-HP (15.3 ± 0.8 g/L) in 48 h with a yield and productivity of 26 % and 0.36 ± 0.02 g/L h, respectively. This shows that the tolerance to and the production of 3-HP differ significantly among the well-known, similar strains of E. coli. The titer and productivity obtained with E. coli W were the highest reported thus far for the biological production of 3-HP from glycerol by E. coli.     

Improved glycerol to ethanol conversion by <Emphasis Type="Italic">E. coli</Emphasis> using a metagenomic fragment isolated from an anaerobic reactor

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD₆₂₀ of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h^?1 OD^?1 L^?1.     

Improving Fatty Acid Availability for Bio-Hydrocarbon Production in Escherichia coli by Metabolic Engineering

Previous studies have demonstrated the feasibility of producing fatty-acid-derived hydrocarbons in Escherichia coli. However, product titers and yields remain low. In this work, we demonstrate new methods for improving fatty acid production by modifying central carbon metabolism and storing fatty acids in triacylglycerol. Based on suggestions from a computational model, we deleted seven genes involved in aerobic respiration, mixed-acid fermentation, and glyoxylate bypass (in the order of cyoA, nuoA, ndh, adhE, dld, pta, and iclR) to modify the central carbon metabolic/regulatory networks. These gene deletions led to increased total fatty acids, which were the highest in the mutants containing five or six gene knockouts. Additionally, when two key enzymes in the fatty acid biosynthesis pathway were over-expressed, we observed further increase in strain △cyoA△adhE△nuoA△ndh△pta△dld, leading to 202 mg/g dry cell weight of total fatty acids, ~250% of that in the wild-type strain. Meanwhile, we successfully introduced a triacylglycerol biosynthesis pathway into E. coli through heterologous expression of wax ester synthase/acyl-coenzyme:diacylglycerol acyltransferase (WS/DGAT) enzymes. The added pathway improved both the amount and fuel quality of the fatty acids. These new metabolic engineering strategies are providing promising directions for future investigation.     

Metabolic engineering of Escherichia coli for the production of succinate from glycerol

Glycerol has become an ideal feedstock for the microbial production of bio-based chemicals due to its abundance, low cost, and high degree of reduction. We have previously reported the pathways and mechanisms for the utilization of glycerol by Escherichia coli in minimal salts medium under microaerobic conditions. Here we capitalize on such results to engineer E. coli for the production of value-added succinate from glycerol. Through metabolic engineering of E. coli metabolism, succinate production was greatly elevated by (1) blocking pathways for the synthesis of competing by-products lactate, ethanol, and acetate and (2) expressing Lactococcus lactis pyruvate carboxylase to drive the generation of succinate from the pyruvate node (as opposed to that of phosphoenolpyruvate). As such, these metabolic engineering strategies coupled cell growth to succinate production because the synthesis of succinate remained as the primary route of NAD+ regeneration. This feature enabled the operation of the succinate pathway in the absence of selective pressure (e.g. antibiotics). Our biocatalysts demonstrated a maximum specific productivity of ～400 mg succinate/gcell/h and a yield of 0.69 g succinate/g glycerol, on par with the use of glucose as a feedstock.     

Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways

A series of Escherichia coli BW25113 mutants with reduced mixed acid fermentation were constructed. Genes ackA-pta, poxB, ldhA, adhE, and pflB encoding acetate kinase, phosphate acetyltransferase, pyruvate oxidase, d-lactate dehydrogenase, acetaldehyde dehydrogenase, and pyruvate formate-lyase, respectively, were deleted successively. When grown under microaerobic condition, the mutants reduced approximately 90% acetate excretion after the deletion of genes ackA-pta and poxB. Production of lactate, ethanol, and formate was also significantly reduced after the deletion of genes ldhA, adhE, and pflB, respectively. The accumulation of biomass and poly(3-hydroxybutyrate) (PHB) were significantly enhanced after deleting the mixed acid fermentation. E. coli mutant BWapld with deletions of ackA-pta, poxB, ldhA, and adhE produced twice the cell dry weight (CDW) and 3.5 times of PHB compared with its wild-type under microaerobic conditions. E. coli mutant BWapl with deletions of ackA-pta, poxB, and ldhA also achieved nearly twice CDW and three times of PHB content in comparison to the wild-type during 48 h static cultivation. Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was observed in the mutants under static cultivation. E. coli mutant BWapld could produce approximately 50 wt.% P(3HB-co-3HV) consisting of 5 mol% of 3-hydroxyvalerate (3HV) under aerobic conditions, when the seed culture was inoculated at an appropriate time. When ackA-pta, poxB, ldhA, adhE, and pflB were deleted, E. coli mutant BWapldf accumulated over 70 wt.% P(3HB-co-3HV) consisting of 8 mol% 3HV under aerobic conditions.     

Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol to Ethanol

Based on elementary mode analysis, an Escherichia coli strain was designed for efficient conversion of glycerol to ethanol. By using nine gene knockout mutations, the functional space of the central metabolism of E. coli was reduced from over 15,000 possible pathways to a total of 28 glycerol-utilizing pathways that support cell function. Among these pathways are eight aerobic and eight anaerobic pathways that do not support cell growth but convert glycerol into ethanol with a theoretical yield of 0.50 g ethanol/g glycerol. The remaining 12 pathways aerobically coproduce biomass and ethanol from glycerol. The optimal ethanol production depends on the oxygen availability that regulates the two competing pathways for biomass and ethanol production. The coupling between cell growth and ethanol production enabled metabolic evolution of the designed strain through serial dilution that resulted in strains with improved ethanol yields and productivities. In defined medium, the evolved strain can convert 40 g/liter of glycerol to ethanol in 48 h with 90% of the theoretical ethanol yield. The performance of the designed strain is predicted by the property space of remaining elementary modes.With the recent rising prices of fossil fuels, development of alternative renewable fuels, such as biodiesel, has become attractive. However, the increase in biodiesel production generates a surplus of crude glycerol since this compound is an inevitable waste by-product resulting directly from the transesterification of vegetable oils or animal fats. For every 3 mol of biodiesel produced, 1 mol of glycerol (about 5 to 10% weight equivalent of biodiesel) is generated. To maximize the full economic potential of the biodiesel process, it is important to convert crude glycerol into useful chemicals (9, 25).Both chemical conversion and biological conversion of crude glycerol into value-added products have been considered. For instance, chemical conversion based on the etherification of glycerol with either alcohols (methanol or ethanol) or alkenes (isobutene or 2-methylpropene) can produce useful fuels or solvents, or steam reforming of glycerol can result in methanol and hydrogen production (7). Biological conversion utilizes species belonging to the Enterobacteriaceae family, such as Klebsiella pneumoniae (11), Citrobacter freundii (5), Clostridium butyricum (4), and Pantoea agglomerans (3), to convert glycerol to 1,3-propanediol by fermentation.Even though Escherichia coli belongs to the family Enterobacteriaceae, it cannot ferment glycerol due to a lack of the dha regulon encoding glycerol dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT), which constitute the fermentative 1,3-propanediol-producing pathway (18). However, introduction of this pathway from K. pneumoniae into E. coli can facilitate glycerol fermentation (18), presumably because the redox potential is balanced. In fact, it is well documented that a wild-type E. coli strain cannot grow on glycerol anaerobically in defined medium except after addition of specific electron acceptors (18). Such electron acceptors can come from external sources, including nitrate, nitrite, fumarate, dimethyl sulfoxide, or trimethylalamine-N-oxide, or from internal sources through added fermentative pathways, such as the 1,3-propanediol-producing pathway.A recent study suggested the feasibility of fermenting glycerol into fuels and other reduced chemicals by inducing the dormant, native 1,2-propanediol fermentative pathway in E. coli without using external electron acceptors (10, 14). This approach, however, faces several critical challenges, such as low specific growth rates resulting in low chemical productivities and coproduction of unavoidable by-products, such as 1,2-propanediol. The reported specific growth rate appears to limit any practical application since a minimal doubling time of about 17 h results in consumption of only 8 to 10 g/liter glycerol after 110 h (10, 14). In addition, glycerol fermentation apparently worked only when complex components, such as yeast extract, tryptone, or amino acids that are required for biomass synthesis, were added to the medium (10, 14).Here, we took a different approach by employing oxygen as the electron acceptor in well-defined microaerobic growth conditions. We used elementary mode (EM) analysis to rationally design an E. coli cell with minimized metabolic functionality tailored to efficiently convert glycerol to ethanol under these microaerobic growth conditions. EM analysis is a metabolic pathway analysis tool that identifies all pathway options in a metabolic network (16). Knowledge of these pathway options allows rational implementation of only the efficient pathways of interest by removing the inefficient pathways, resulting in a cell with minimal but specialized functionality (20-22, 24). Furthermore, the cell developed is engineered to tightly couple cell growth and ethanol production. This unique characteristic facilitates metabolic evolution of the minimal cell to improve ethanol productivity because faster-growing cells also produce ethanol at a higher rate. We demonstrate here that the performance of the designed strain falls into the range defined by the EMs that are possible.