Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli
© Mazumdar et al.; licensee BioMed Central Ltd. 2013
Received: 27 September 2012
Accepted: 20 January 2013
Published: 25 January 2013
Due to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid (L-lactate).
Escherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity.
This study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity.
A shared metabolic feature of the anaerobic and microaerobic utilization of glycerol in E. coli is the generation of ethanol as the primary product and the negligible production of lactic acid (lactate) [5–7]. However, we have recently reported the engineering of this bacterium for microaerobic production of D-lactate from glycerol in a defined minimal medium . Lactate and its derivatives have many applications in the food, pharmaceutical, and polymer industries [16, 17]. An example is polylactic acid, a renewable, biodegradable, and environmentally friendly polymer produced from controlled ratios of D- and L-lactate . Because of the importance of using pure enantiomers in such applications, biological processes have the advantage over chemical means of producing chirally pure lactate from inexpensive media containing only the carbon source and mineral salts . While lactic acid bacteria have been traditionally used in the production of D- and L-lactate from carbohydrate-rich feedstocks, several studies have recently reported alternative biocatalysts such as E. coli[16, 17], many of which are engineered to produce L-lactate from sugar feedstocks [20–23].
Unlike the aforementioned reports (i.e. use of carbohydrates), our laboratory has focused on the use of glycerol as a carbon source for the production of chemicals with high optical and chemical purity. As such, this manuscript focuses on the metabolic engineering of E. coli for the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported prior to our work. The engineered strains hold great promise for the conversion of low-value glycerol streams present in the current biofuels industries to a higher-valued product, L-lactate.
Replacement of E. coli’s D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase and disruption of the methylglyoxal bypass
Glycerol consumption, product synthesis, and carbon recovery in cell mass and fermentation products during the microaerobic utilization of glycerol in minimal medium by wild-type and engineered strains a
Glycerol consumed (g/L)
Product synthesized (g/L)
Strains engineered for the production of D-lactate
Strains engineered for the production of L-lactate
Functional characterization of constructs used in the overexpression of glycerol utilization and L-lactate synthesis enzymes
Activity (μmol/mg protein/min)a
0.187 ± 0.005
0.669 ± 0.004
Aerobic glycerol-3-phosphate dehydrogenase
0.017 ± 0.001
0.027 ± 0.002
0.049 ± 0.002
0.39 ± 0.02
0.005 ± 0.001
0.019 ± 0.002
0.136 ± 0.005
0.68 ± 0.06
Overexpression of glycerol-utilization and L-lactate synthesis pathways and elimination of the endogenous pathway for L-lactate utilization
Although strains LA19 and LA20 produced L-lactate at high chemical and chiral purity, the kinetics of glycerol utilization and lactate synthesis, including lactate titer and yield, were inferior to that of the LA01 and LA02 parental strains (compare panels A and B in Figure 2; Table 1). Since we have previously shown that the conversion of glycerol to D-lactate can be accelerated by amplifying either glycerol-utilization or lactate-synthesis pathways , we investigated whether similar strategies could be implemented in the production of L-lactate.
Two primary routes can mediate the conversion of glycerol to the common intermediate, dihydroxyacetone (DHAP) under microaerobic conditions  (Figure 1). A fermentative pathway converts glycerol to dihydroxyacetone (DHA) via glycerol dehydrogenase (gldA) and then to DHAP through the action of DHA kinase (dhaKLM). The alternative route is a respiratory/aerobic pathway composed of the enzymes glycerol kinase (glpK) and glycerol-3-phosphate (G3P) dehydrogenase (glpD) which mediates the conversion of glycerol to G3P and subsequently to DHAP, respectively. Overexpression of either one of the pathways in LA19 (0.3 and 0.33 g/L/h for fermentative and respiratory routes, respectively) and LA20 (0.28 and 0.38 g/L/h for fermentative and respiratory routes, respectively) led to faster utilization of glycerol and L-lactate synthesis, although the respiratory pathway led to higher L-lactate titers and yields (Table 1, 2). Coupling of glycerol-3-phosphate oxidation and oxygen reduction via the quinine pools [28, 29] likely results in the preferential synthesis of L-lactate due to the fact that the overall conversion of glycerol to lactate becomes a redox balanced pathway. In addition, ATP would be generated by both substrate-level phosphorylation and the respiratory chain (see Figure 1 and Discussion).
Another limiting factor for lactate synthesis in strains LA19 and LA20 could be insufficient levels of L-lactate dehydrogenase due to less expression from the chromosomal copy of S. bovis ldh as opposed to plasmid overexpression. Thus, expression of ldh from a plasmid could alleviate this limitation and lead to an increase in the fraction of carbon diverted towards the synthesis of L-lactate (increasing L-lactate yield) and/or the flux of the glycerol-to- L-lactate pathway (increasing the rate of L-lactate production). This strategy led to a slight increase in the production of L-lactate in LA20 [pZSldh] (Table 1), which was arguably caused by the 5-fold increase in the activity of L-LDH (Table 2). In contrast, overexpression of L-LDH had no beneficial effect on lactate production or glycerol utilization in strain LA19 [pZSldh] (Table 1). Thus, plasmid overexpression of S. bovis ldh was not deemed more beneficial than that of the chromosomal copy and not explored further.
Of note, strain LA19 (ΔpflB ΔfrdA ΔmgsA ΔldhAldh+) and its parent and derivatives produced much higher concentrations of acetate than that observed in the LA20 (Δpta ΔadhE ΔfrdA ΔmgsA ΔldhAldh+) strain and its parent and derivatives (Table 1). While PFL is the primary route for pyruvate conversion to acetyl-CoA during the microaerobic utilization of glycerol, low levels of acetyl-CoA and subsequently acetate could still be formed in the LA19 lineage via leakiness of the primarily aerobic pyruvate dehydrogenase complex (aceEF and lpdA, Figure 1) . As acetate formation in the LA20 lineage is directly blocked by a pta deletion, lower acetate levels would be expected. Increased acetate formation in the LA19 lineage could also explain the differential growth observed between LA06 (i.e. ΔpflB, pta+ etc.) and LA07 (i.e. pflB+, Δpta). As these strains are deleted for endogenous ldhA, they cannot readily synthesize any common fermentative product to achieve redox balance and allow continued ATP production. In this context, the small increases in acetate levels seen in the LA06 would be critical for growth as acetate formation results in 2 ATP molecules per glycerol consumed via substrate level phosphorylation (Figure 1). Only when the higher glycerol utilization and subsequent L-lactate synthesis were achieved with the more optimal expression of S. bovis ldh from the chromosome (as opposed to from a plasmid) did the growth between the LA19 and LA20 and direct derivatives become similar (Figure 2 and Table 1). As the LA20 lineage was deemed better than that of LA19 and previous work by us has shown no additional benefit of using the pflB deletion in conjunction with just directly blocking the competing fermentative products (data not shown) we choose to use LA20 as our platform for further metabolic engineering.
Production of L-lactate at high concentrations from crude glycerol
L-lactate production from sugars can be achieved using native lactic acid bacteria but are constrained by the requirements for complex nutrients and exhibit limitations in both product selectivity and enantiomeric purity [16, 17]. To overcome these issues, bacteria and yeasts have been engineered to produce L-lactate as the primary product of carbohydrate fermentations [16, 17, 20–23]. However, the production of L-lactate from glycerol has not been reported. The work conducted here focuses on the metabolic engineering of E. coli for the microaerobic production of L-lactate, at high chemical (97%) and optical (99.9%) purities, from glycerol in defined minimal salts medium. Using LA20ΔlldD [pZSglpKglpD], 50 g/liter of L-lactate were produced in 84 hours at a yield of 0.90 g L-lactate/g glycerol (Figure 5) with a yield close to 93% of the theoretical maximum (0.967 wt/wt) when calculated from equation 2 below. Besides providing a high yield and productivity, the resulting biocatalyst can also utilize crude glycerol as carbon source, which has become an abundant and inexpensive feedstock due to being a by-product of the current biofuel industries .
From equations 2 and 3 it then becomes apparent that the synthesis of L-lactate from glycerol can generate up to two molecules of ATP per molecule of L-lactate produced. Overall, this high ATP yield explains why using the later stages of the Embden-Meyerhof-Parnas pathway with overexpression of the respiratory GlpK-GlpD pathway in LA20 was beneficial (as opposed to the use of the MG route).
Given the beneficial nature of the engineered glycerol-to-L-lactate pathway (i.e. redox balanced and ATP generating), we expect that the future use of metabolic evolution approaches will lead to the selection of even more productive biocatalysts. Similar techniques have been successfully implemented in E. coli for the efficient production of biofuels and other products [34–36]. Process-based modifications such as fed-batch cultivations and high-density cultures are also envisioned to further improve the volumetric rates of L-lactate production.
The present study demonstrates the conversion of glycerol to L-lactate, a microbial process that had not been reported to date prior to this study. The engineered biocatalyst produced L-lactate from glycerol in a defined minimal salts medium at high chemical and optical purity. The high yields and productivities achieved with the use of crude glycerol as carbon source, which has become an abundant and inexpensive feedstock, demonstrate that low-value glycerol streams from the current biofuels industries can be efficiently converted to higher value products such as L-lactate.
Strains, plasmids, and genetic methods
Strains, plasmids and primers used in this study
F- λ- ilvG- rfb-50 rph-1
MG1655 ΔpflB::FRT ΔfrdA::FRT-Kan-FRT; sequential deletion of pflB and frdA in MG1655
MG1655 Δpta::FRT ΔadhE::FRT ΔfrdA::FRT-Kan-FRT; sequential deletion of pta, adhE and frdA in MG1655
LA01 ΔmgsA::FRT ΔldhA::ldh
LA02 ΔmgsA::FRT ΔldhA::ldh
LA01 ΔmgsA::FRT ΔldhA::ldh ΔlldD::FRT
LA02 ΔmgsA::FRT ΔldhA::ldh ΔlldD::FRT
reppSC101ts ApR CmR cI857 l PR flp+
Blank plasmid created by removing C. freundii dhaKL from pZSKLcf and self-ligating the plasmid (tetR, oriR SC101*, cat)
f1(+) ori lacZ α of pBluescript II (SK+) mobRP4, oriR6K,SacB and AmpR
E. coli dhaKLM and gldA under control of PLtetO-1 (tetR, oriR SC101*, cat)
E. coli glpK and glpD under control of PLtetO-1 (tetR, oriR SC101*, cat)
S. bovis ldh under control of PLtetO-1 (tetR, oriR SC101*, cat)
Gene overexpression was achieved by cloning the desired gene(s) in a low-copy vector as previously reported  (Table 3). Plasmid pZSldh was constructed as follows. The ldh gene from S. bovis was PCR amplified from plasmid pVALDH1  using c-ldh primers (Table 3). The resulting PCR product was cloned within the Kpn I and Mlu I sites of pZSKLMgldA  using In-Fusion PCR cloning (Clontech Laboratories, Inc., Mountain View, CA). PCR was performed using Pfu turbo DNA polymerase (Stratagene, CA, USA) under standard conditions described by the supplier. The ligated products were used to transform E. coli DH5αT1 (Invitrogen, Carlsbad, CA). Positive clones were screened by plasmid isolation and restriction digestion.
Standard recombinant DNA procedures were used for gene cloning, plasmid isolation, and electroporation. Manufacturer protocols and standard methods [37, 43] were followed for DNA purification (Qiagen, Valencia, CA), restriction endonuclease digestion (New England Biolabs, Ipswich, MA), and DNA amplification (Stratagene, La Jolla, CA and Invitrogen, Carlsbad, CA). The strains were kept in 32.5% glycerol stocks at −80°C. Plates were prepared using LB medium containing 1.5% agar, and appropriate antibiotics were included at the following concentrations: ampicillin (50 μg/ml), kanamycin (50 μg/ml), chloramphenicol (12.5 μg/ml), and tetracycline (3.33 μg/ml).
Culture medium and cultivation conditions
Unless otherwise stated, all fermentations were conducted using the minimal medium designed by Neidhardt et al. with Na2HPO4 in place of K2HPO4 and supplemented with 20 g/liter glycerol (unless otherwise specified), 5 μM sodium selenite, 3.96 mM Na2HPO4, 5 mM (NH4)2SO4, and 30 mM NH4Cl. Chemicals were obtained from Fisher Scientific (Pittsburgh, PA) and Sigma-Aldrich Co. (St Louis, MO), except crude glycerol, which was provided by Renewable Energy Group, Inc. (Ames, IA). Crude glycerol had the following composition (wt/wt%): glycerol (83.3), methanol (0.01), water (10.0), fatty acids (0.04), salt (6.63), and ash (6.6). The pH was 6.38 and the density was 1.26 g/ml.
Fermentations in shake flasks were performed in 25 ml Pyrex Erlenmeyer flasks (narrow mouth/heavy duty rim, Corning Inc., Corning, NY) filled with 15 ml of 1X MOPS minimal media supplemented with appropriate antibiotics or inducers when needed at the following concentrations: ampicillin (50 μg/ml), kanamycin (50 μg/ml), chloramphenicol (12.5 μg/ml), tetracycline (3.33 μg/ml), and anhydrotetracycline (100 ng/ml). Unless otherwise stated, calcium carbonate (5% wt/wt) was used in all the fermentation flasks to buffer the pH. The flasks (with foam plugs filling the necks) were incubated at 37°C and 200 rpm in an NBS C24 Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, NJ). The fermentations were run for 36 hours (unless otherwise stated) at which time the supernatant was collected, the pH measured (UB-10, Denver Instruments Co., Arvada, CO), the optical density taken (Thermo Spectronic Genesys 20, 4001/4, MA, USA), and when necessary cell pellets collected for enzyme activity assays. To determine the optical densities of the cultures in the presence of calcium carbonate, the cultures were allowed to briefly sit in which time the calcium carbonate quickly settled to the bottom.
Prior to use, the cultures (stored as glycerol stocks at −80°C) were streaked onto LB plates and incubated overnight at 37°C. Three colonies were used to inoculate 25-ml flasks containing 5 ml of minimal medium supplemented with 10 g/liter of glycerol, 10 g/liter tryptone, and 5 g/liter yeast extract. The flasks were incubated at 37°C and 150 rpm in an NBS C24 Benchtop Incubator Shaker until an OD550 of ~0.7 was reached. An appropriate volume of this actively growing pre-culture was centrifuged, and the pellet was washed and used to inoculate 15 ml of medium in shake flasks (see above) with a target initial optical density at 550 nm of 0.05.
The concentration of cell mass, glycerol, organic acids, and ethanol were measured as previously described [45, 46]. The enantiomeric purity of lactate was determined enzymatically as previously reported . The reaction mixture (3 ml) for L-lactate determination contained 0.92 ml hydrazine/glycine buffer (0.6 M glycine and 0.5 M hydrazine; pH 9.2), 55 U L-lactate dehydrogenase, 5 mg NAD, and 200 μL of the fermentation sample of interest. D-lactate was measured in a similar mixture by replacing L-lactate dehydrogenase with 15 U of D-lactate dehydrogenase. After addition of the sample, the reaction mixture was incubated at 25°C for 3 hours after which the absorbance at 340 nm was used as a measure of the concentration of D- or L-lactate present.
Cell harvesting and preparation of crude cell extracts for enzyme assays was conducted as described elsewhere [5, 7]. Absorbance changes for all assays were monitored in a Biomate 5 spectrophotometer (Thermo Scientific, MA, USA). The linearity of reactions (protein concentration and time) was established for all assays and the nonenzymatic rates were subtracted from the observed initial reaction rates. Enzymatic activities are reported as μmol of substrate per minute per mg of cell protein and represent averages for at least three cell preparations. A protein content of 55% (wt/wt) for E. coli cells was assumed in these calculations.
Glycerol kinase and aerobic-glycerol-3-phosphate dehydrogenase activities were assayed as reported previously . Details of the assay can be found elsewhere . The activity of glycerol dehydrogenase in the oxidation of glycerol was measured as previously described  with potassium carbonate at pH 9.5 as the buffer. PEP-dependent dihydroxyacetone kinase activity was assayed as previously reported . D-lactate dehydrogenase activity was determined by following the NADH-dependent reduction of pyruvate at 340 nm and 25°C in a 1 ml reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 30 mM sodium pyruvate, 0.33 mM NADH, and 50 μL crude cell extract . The activity of L-lactate dehydrogenase (encoded by S. bovis ldh) was determined as described above for D-lactate dehydrogenase but adding fructose 1,6-bisphosphate, an allosteric activator of S. bovis L-LDH , to the mixture at a final concentration of 1.2 mM.
Calculation of fermentation parameters
Data from cell growth, glycerol consumption, and product synthesis were used to calculate volumetric (g/liter/h) and specific rates (g/g cell mass/h) and product yields (g/g glycerol) as previously described [5, 11].
This work was supported by grants from the U.S. National Science Foundation (CBET-0645188) and the National Research Initiative of the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service (2005-35504-16698). We thank H. Mori and T. R. Whitehead for providing research materials, C. Rivera, S. Doneske, and S. S. Yazdani for assistance with genetic methods, Alfredo Martinez Jimenez for assistance in the quantification of oxygen transfer, and Paul Campbell for fruitful discussions.
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