Engineering Escherichia coli K12 MG1655 to use starch
© Rosales-Colunga and Martínez-Antonio; licensee BioMed Central Ltd. 2014
Received: 30 September 2013
Accepted: 11 May 2014
Published: 21 May 2014
To attain a sustainable bioeconomy, fuel, or valuable product, production must use biomass as substrate. Starch is one of the most abundant biomass resources and is present as waste or as a food and agroindustry by-product. Unfortunately, Escherichia coli, one of the most widely used microorganisms in biotechnological processes, cannot use starch as a carbon source.
We engineered an E. coli strain capable of using starch as a substrate. The genetic design employed the native capability of the bacterium to use maltodextrins as a carbon source plus expression and secretion of its endogenous α-amylase, AmyA, in an adapted background. Biomass production improved using 35% dissolved oxygen and pH 7.2 in a controlled bioreactor.
The engineered E. coli strain can use starch from the milieu and open the possibility of optimize the process to use agroindustrial wastes to produce biofuels and other valuable chemicals.
The development of biomass-based processes is key to the establishment of sustainable and petroleum-independent industry . Therefore, the production of valuable products, such as biofuels, lactic acid, and ferulic acid, is currently based on the use of biomass to close the CO2 cycle. In this cycle, the CO2 is integrated into biomass, released, and reincorporated again into biomass, which differs from the use of fossil counterparts.
Biomass resources range from lignocellulosic sources to grains that are usually employed for human consumption. Despite the great amount of biomass available in crops, their use as human food raises concern for their role in biotechnological processes. Thus, discovering alternatives sources is critical. Many of the processes under development focus on hydrolysis of lignocellulosic materials for microbial-biomass production. However, there are many other sources of biomass available, including agroindustrial by-products or wastes such as cheese whey [2, 3], olive mill waste-water , cassava bagasse, citrus-processing residues , and manure . From these biomass sources, there is the potential to obtain valuable products.
One of the main biomass components is starch . The predominant natural sources of starch are maize, potato, wheat, rice , and other diverse tubercles that are widely used in food-processing industries and generate a large amount of waste. For instance, potato pulp, which is a by-product generated from potato processing, usually contains a considerable amount of starch and fibers, which could represent an excellent source for the production of valuable products, such as lactic acid . Furthermore, in plants and particularly tubercles, almost every carbon storage tissue has elevated starch content. These organic materials are largely neglected as potential starch sources.
To take advantage of starch-rich bio-resources, it is necessary to develop microbes capable of hydrolyzing starch and utilizing their products. To achieve this goal, utilization and adaptation of hydrolytic enzymes is necessary. In industry, hydrolysis of these biomass materials requires enzymatic and/or acid hydrolysis pre-treatment prior to fermentation. A bacterial strain that develops both hydrolysis and fermentation would be extremely valuable.
The bacterium E. coli is widely used as a model organism both in research and industrial processes, and its genome encodes a cytosolic α-amylase, AmyA. It is of great interest to secrete this enzyme in the milieu to determine its exogenous hydrolytic capacity. Among secretion signal peptides, the FhuD signal shows high secretory activity. This activity could be due to its capacity to use the two secretory pathway systems present in E. coli: Tat and Sec .
For starch hydrolysis, α-amylase should be expressed by the bacterium at basal levels to immediately begin starch hydrolysis in the milieu. Ideally, the resultant products of starch degradation, maltose and maltodextrins, should further activate AmyA expression. In E. coli, expression of native genes related to maltose/maltodextrin catabolism and their transport is controlled by 5 operons that include 10 genes. These consist of ABC transporters and catabolic enzymes regulated by the transcription factor MalT [11–13]. However, there is no evidence that the amyA native gene is part of this system, and it is not regulated by MalT. Genes regulated by MalT show basal expression levels when maltose/maltodextrins are not present . This expression is augmented in the presence of maltose/maltodextrins that function as co-activators of MalT, and maltodextrins of up to seven units can be transported into the cytosol to sustain E. coli growth .
Strain development to increase the efficiency of microorganisms has been an important objective of the biotechnology industry for decades . Among existing methods that improve strain performance, the selection of spontaneous adaptive mutants is a simple, and in most of cases, effective technique. Based on reports of catabolic adaptive mutants, the most common mechanisms described are conversion of an induced system to a constitutive or de-repressed system [16, 17], and increasing permeability and substrate uptake [16, 18].
In this study, we selected adaptive mutants to grow on starch-containing media and use a synthetic biology approach based on BioBricks technology, which employs restriction-site standardized genetic parts, to develop an E. coli strain able to consume starch. Our aim was to engineer an E. coli strain that can serve as a starting point to be used as microbial platform to use agroindustrial wastes to produce biofuels and other valuable compounds.
Results and discussion
Adapted E. coli strain selection
Shibuya et al. reported similar growth profiles for other adapted strains as well as extracellular amylolytic activity from the adapted strains. However, in the present work, the adapted strains did not show amylolytic activity because no starch degradation was observed (Figure 1). Thus, the observed growth could be due to better use of small maltodextrins or additional contents in the commercial starch employed, the presence of which cannot be detected by the method used here. Indeed, the initial culture media was analyzed by high-performance liquid chromatography (HPLC). The results indicated a mix of maltodextrins of 3–5 glucose units and trace amounts of glucose, fructose, maltose, and sucrose, in addition to the starch content. Because similar biomass yields were obtained from the two adapted strains, we used the WTa6 strain in all subsequent experiments.
Design and expression of α-amylase
Because the adapted strains did not show amylolytic activities, it is possible that the expression of exogenous α-amylase might improve starch utilization by the adapted strain and further increase the yield of biomass or any desired product derived from starch. The E. coli genome encodes the cytosolic α-amylase, AmyA. The reported substrates for this enzyme are maltodextrins with least six glucose units, amylose, and starch. Amylopectin is used less effectively .
To increase the uptake of carbon sources derived from starch, we engineered secretion of the AmyA enzyme using the FhuD signal peptide. By analyzing published microarray studies, we found that among the genes that are regulated by MalT, malE shows the highest response [22, 23]. Therefore, we selected the malE promoter region to be used in our synthetic genetic construction. In this way, α-amylase expression and secretion could be coupled to the maltose/maltodextrin system. The plasmid pAM encodes the amyA construct in the plasmid pSB1A2 (see Methods section).
α-amylase secretion and bacterial growth
Improved biomass yield from starch
These data highlight the potential of this strain to use starch to yield valuable products.
In this study, we developed an E. coli strain capable of using extracellular starch as the sole carbon source. This was achieved by following a synthetic biology framework. The process takes advantage of the native capability of E. coli to use maltodextrins as carbon sources and a cytoplasmic amylase that was engineered to alter its transcription and secretion. The genetic construct generated a functional amylase with a signal peptide that permitted its secretion to the milieu. α-amylase expression and secretion allowed the bacterium to grow actively using starch as the sole carbon source. The starch degradation products activated α-amylase gene transcription, constituting a positive feedback circuit for amylase expression. Adapted strains grown on starch plates could be identified and selected by their ability to form colonies faster than WT. The use of these adapted strains as a background to harbor the engineered cytoplasmic amylase improved biomass yield using starch as substrate. Under controlled conditions (pH 7.2 and 35% DO), the biomass production of Amy6 was further increased. This work opens the potential to optimize the process to finally use this strain as a platform to produce added value products using starch as substrate.
Strains, culture media, and growth conditions
Strains and plasmids
Genotype or description
Escherichia coli K-12 MG1655
WT, starch-adapted strains
WT with the backbone plasmid pSB1A2
WT with pAM
WTa6 with pAM
WT with amyA deleted
BioBricks backbone plasmid
BioBricks backbone plasmid
pSB1A2 with engineered amyA
Strains were grown routinely on LB plates. Pre-inoculants were grown in LB overnight, centrifuged at 10,000 rpm, and resuspended in M9 minimal media without a carbon source. They were inoculated in 125 mL or 250 mL baffled flasks with M9 plus carbon sources (5 g/L). Pre-inoculants for biomass yield from starch were obtained using M9 with 5 g/L maltose (Sigma) or soluble starch (Bio Basic Canada Inc.). Samples were taken periodically to evaluate growth at OD600, as well as for starch or sugar determination. Batch cultures were performed in a 3-L bioreactor (Applikon, The Netherlands) with a working volume of 1 L. The pH and dissolved oxygen (DO) were monitored online using autoclavable electrodes (AppliSense, The Netherlands). These parameters were automatically controlled using PID control with the My controlTM controller (Applikon) using 2.5 N NaOH and HCl solutions for the pH control and air for DO. The set points were 7.2 and 35% for pH and DO, respectively. Cultures were maintained at 37°C and stirred at 200 rpm with two six-blade Rushton turbines. BioXpert software (Applikon) was used for data acquisition.
Plate experiments were performed in M9 plates with 5 g/L of soluble starch. All assays were performed at 37°C. Carbenicillin (Sigma) at a final concentration of 100 mg/L was used in all cultures harboring the pAM plasmid.
Selection of adapted strains
Two hundred microliters WT E. coli grown overnight were spread on M9/ starch plates and incubated at 37°C until colonies appeared. The resulting colonies were cultured again on fresh M9/ starch plates and incubated at 37°C to observe their growth. The strains with the fastest growth (i.e., colony formation) were further tested in M9/starch liquid media. A colony sample of each of the selected strains was taken and inoculated in 10 mL of M9/starch. The OD600 was monitored to select the fastest growing strains in liquid media.
Genetic construction for starch hydrolysis
BioBricks and primers used to obtain a genetic construct for starch hydrolysis
Description or Sequence (5'-3')
Artificial double-transcriptional terminator
Signal peptide from fhuD, obtained by PCR with the primers spF and spR
Promoter from malE, obtained by PCR with the primers prF and prR
amyA gene, obtained by PCR with the primers amF and amR
GTTTCTTCGAATTC GCGGCCGCTTCTAGA TGAGCGGCTTACCTCTTAT
GTTTCTTCGAATTC GCGGCCGCTTCTAGA GTGGCTTAAATCCTCCACCCC
GTTTCTTCGAATTC GCGGCCGCTTCTAGA CGTAATCCCACGCTGTTACA
Each genetic biopart was obtained by PCR and assembled by rounds of digestion with specific restriction enzymes (Fermentas) and ligations (T4 DNA ligase, Thermo Scientific). Briefly: 1) malEp was cut with EcoRI and SpeI, at the same time the vector pSB1A2, containing the RBS BBa_B0030 was cut with EcoRI and XbaI, and then ligated to obtain the “malEp-RBS” construction in the vector pSB1A2. 2) The same was done with amyA and the double terminator of the transcription BBa_B1002 to get the “amyA-double terminator” construction in the vector pSB1AK3. 3) The signal peptide fhuDsp was cut with XbaI and PstI and the malEp-RBS construction (in the vector pSB1A2 mentioned above) was cut with SpeI and PstI; these fragments were ligated to obtain the “malEp-RBS-fhuDsp” construction in the vector pSB1A2. 4) Finally, this last construction was cut with SpeI and PstI, and ligated with the digestion of amyA-double terminator to obtain the pAM plasmid. The final genetic construction encloses: the malE promoter, a RBS, the fhuD signal peptide, the alpha amylase gene amyA and the double terminator of the transcription in the backbone vector pSB1A2. Every construction was confirmed by PCR and restriction enzyme digestion analysis and the final construction by DNA sequencing.
Amylase expression assays
SDS-PAGE was performed using 10% polyacrylamide in a Mini Protean Tetra Cell (BioRad) and stained with Coomassie blue R250. To determine the amylolytic activity of plates, starch-containing plates were stained with 1.5 mL of a 1:5 dilution of lugol solution (7.21, 5.17, and 86.6 g/L of I2, KI, and ethanol, respectively; Karal, México).
Quantitative starch determination
Starch content was determined by its ability to complex iodine . First, the maximum peak absorbance of soluble starch was determined by scanning a starch sample with lugol from 200 to 800 nm. Maximum absorbance was observed at 570 nm. Next, a calibration curve of starch was performed at 570 nm. To quantify starch, samples were centrifuged at 10,000 rpm for 5 minutes. Ten microliters of lugol (1:10 dilution) was added to the supernatant, and the absorbance was immediately measured at 570 nm (Beckman DU 640 Spectrophotometer).
Glucose, maltose, and maltodextrin determination
Sugar content was determined by 3, 5-dinitrosalicylic acid (DNS) assays. Briefly, 0.5 mL of samples were mixed with 0.5 mL DNS reagent [10 g/L DNS (Sigma), 16 g/L NaOH (Karal, México), 300 g/L sodium potassium tartrate (Sigma)], boiled 5 min, and cooled on ice. Next, the mixture was diluted (1:5 dilution) and the absorbance at 540 nm was measured. The absorbance was compared with the calibration curve of each sugar. Maltodextrins in the culture media were analyzed using the HPLC system Agilent HP 1200 series (Agilent Technologies) equipped with Zorbax carbohydrate column (4.6 × 150 mm 5-micron PN 843300–908; Agilent Technologies) and a refraction index detector (RID). The mobile phase of 75:25 (v/v) acetonitrile: water was used and run using an isocratic gradient at a flow rate of 1.4 mL/min and 30°C.
This work was partially funded by CONACYT through a PROINNOVA grant. LMRC thanks CONACyT for a postdoctoral fellowship award. The authors acknowledge the technical assistance of Susana Ruíz Castro, Pablo Israel Vargas, MSc. Arlette Bohórquez Hernández, and Dr. Guillermo Pastor Palacios. We thank Dr. John Paul Délano-Frier for English corrections.
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