Construction of a grpE-based plasmid addiction system in Escherichia coli and its application in phloroglucinol biosynthesis.

AIM: Biotechnical processes in Escherichia coli often operate with artificial plasmids. However, these bioprocesses frequently encounter plasmid loss. To ensure stable expression of heterologous genes in E. coli BL21(DE3), a novel plasmid addiction system (PAS) was developed. METHODS AND RESULTS: This PAS employed an essential gene grpE encoding a cochaperone in the DnaK-DnaJ-GrpE chaperone system as the selection marker, which represented a chromosomal ΔgrpE mutant harboring episomal expression plasmids that carry supplementary grpE alleles to restore the deficiency. To demonstrate the feasibility of this system, it was implemented in phloroglucinol (PG) biosynthesis, manifesting improved host tolerance to PG and increased PG production. Specifically, PG titer significantly improved from 0.78 ± 0.02 to 1.34 ± 0.04 g l-1, representing a 71.8% increase in shake-flask fermentation. In fed-batch fermentation, the titer increased from 3.71 ± 0.11 to 4.54 ± 0.10 g l-1, showing a 22.4% increase. RNA sequencing and transcriptome analysis revealed that the improvements were attributed to grpE overexpression and upregulation of various protective chaperones and the biotin acetyl-CoA carboxylase ligase coding gene birA. CONCLUSION: This novel PAS could be regarded as a typical example of nonanabolite- and nonmetabolite-related PAS. It effectively promoted plasmid maintenance in the host, improved tolerance to PG, and increased the titer of this compound.

Production of Optically Pure (S)-3-Hydroxy-γ-butyrolactone from d-Xylose Using Engineered Escherichia coli.

Biocatalysis has advantages in asymmetric synthesis due to the excellent stereoselectivity of enzymes. The present study established an efficient biosynthesis pathway for optically pure (S)-3-hydroxy-γ-butyrolactone [(S)-3HγBL] production using engineered Escherichia coli. We mimicked the 1,2,4-butanetriol biosynthesis route and constructed a five-step pathway consisting of d-xylose dehydrogenase, d-xylonolactonase, d-xylonate dehydratase, 2-keto acid decarboxylase, and aldehyde dehydrogenase. The engineered strain harboring the five enzymes could convert d-xylose to 3HγBL with glycerol as the carbon source. Stereochemical analysis by chiral GC proved that the microbially synthesized product was a single isomer, and the enantiomeric excess (ee) value reached 99.3%. (S)-3HγBL production was further enhanced by disrupting the branched pathways responsible for d-xylose uptake and intermediate reduction. Fed-batch fermentation of the best engineered strain showed the highest (S)-3HγBL titer of 3.5 g/L. The volumetric productivity and molar yield of (S)-3HγBL on d-xylose reached 50.6 mg/(L·h) and 52.1%, respectively. The final fermentation product was extracted, purified, and confirmed by NMR. This process utilized renewable d-xylose as the feedstock and offered an alternative approach for the production of the valuable chemical.

Preparation and identification of a novel peptide with high antioxidant activity from corn gluten meal.

The antioxidant activity of corn peptides is related to their molecular weight and structure. Corn gluten meal (CGM) was hydrolyzed using a combination of Alcalase, Flavorzyme and Protamex, and the hydrolysates were subjected to antioxidant activity analysis after further fractionation. Corn peptides with molecular weights less than 1 kDa (CPP1) exhibited excellent antioxidant activity. A novel peptide, Arg-Tyr-Leu-Leu (RYLL), was identified from CPP1. RYLL displayed preferable scavenging capacities for ABTS radicals and DPPH radicals, with IC50 values of 0.122 mg/ml and 0.180 mg/ml, respectively. Based on quantum calculations, RYLL had multiple antioxidant active sites, and tyrosine was the main active site due to the highest energy of the highest occupied molecular orbit (HOMO). Moreover, the simple peptide structure and hydrogen bond network of RYLL contributed to the exposure of the active site. This study elucidated the antioxidant mechanism of corn peptides, which could provide an understanding for CGM hydrolysates as natural antioxidants.

Modification of Fatty Acid Composition of Escherichia coli by Co-Expression of Fatty Acid Desaturase and Thioesterase from Arabidopsis thaliana.

Fatty acid composition has an important influence on the fluidity of biological membranes, which is a key factor for the survival of Escherichia coli. With the aim to modify fatty acid composition in this experimentally friendly microorganism, the AtFab2 gene, encoding the Arabidopsis thaliana fatty acid desaturase, was expressed separately and jointly with AtFatA, a fatty acid thioesterase of the same plant origin. The expression of ATFab2 desaturase resulted in an enhancement of cis-vaccenic acid (18:1Δ11) contents, while amounts of palmitioleic acid (16:1Δ9) accumulated by E. coli were increased by 130% for the expression of the AtFatA thioesterase. In the final engineered strain co-expressing AtFab2 and AtFatA, the percentage of palmitic acid (16:0), the most abundant saturated fatty acid found in E. coli, was reduced to 29.9% and the ratio of unsaturated fatty acid to saturated fatty acid reached 2:1. Free fatty acids accounted for about 40% of total fatty acid profiles in the recombinant strain expressing both two genes, and the unsaturated fatty acid contents reached nearly 75% in the free fatty acid profiles. The increase of unsaturated fatty acid level might provide some implication for the construction of cold tolerant strains.

Debottlenecking the biological hydrogen production pathway of dark fermentation: insight into the impact of strain improvement.

Confronted with the exhaustion of the earth's fossil fuel reservoirs, bio-based process to produce renewable energy is receiving significant interest. Hydrogen is considered as an attractive energy carrier that can replace fossil fuels in the future mainly due to its high energy content, recyclability and environment-friendly nature. Biological hydrogen production from renewable biomass or waste materials by dark fermentation is a promising alternative to conventional routes since it is energy-saving and reduces environmental pollution. However, the current yield and evolution rate of fermentative hydrogen production are still low. Strain improvement of the microorganisms employed for hydrogen production is required to make the process competitive with traditional production methods. The present review summarizes recent progresses on the screening for highly efficient hydrogen-producing strains using various strategies. As the metabolic pathways for fermentative hydrogen production have been largely resolved, it is now possible to engineer the hydrogen-producing strains by rational design. The hydrogen yields and production rates by different genetically modified microorganisms are discussed. The key limitations and challenges faced in present studies are also proposed. We hope that this review can provide useful information for scientists in the field of fermentative hydrogen production.

Identification of Two Novel Fluorinases From Amycolatopsis sp. CA-128772 and Methanosaeta sp. PtaU1.Bin055 and a Mutant With Improved Catalytic Efficiency With Native Substrate.

Fluoride plays an important role in the fields of materials and medicine. Compared with chemical synthesis, fluorinases are natural catalysts with more application potential, which provide a green and effective way to obtain organofluorine. However, the application of fluorinases is limited by certain factors, such as the limited number of enzymes and their low activity. In this work, two new fluorinases from Amycolatopsis sp. CA-128772 and Methanosaeta sp. PtaU1.Bin055 were identified by gene mining and named Fam and Fme, respectively. The activities of these two enzymes were reported for the first time, and Fme showed good thermal stability, which was different from the reported fluorinases. In addition, the activity toward natural substrate of Fam was improved by site-directed mutagenesis, the catalytic efficiency (k cat /K m ) of the best mutant containing two amino acid substitutions (T72A and S164G) toward the substrate S-adenosyl-L-methionine was improved by 2.2-fold compared to the wild-type. Structural modeling analysis revealed that the main reason for the increased enzyme activity might be the formation of a new substrate channel. Experimental evidence suggests that the substrate channel may indeed play a key role in regulating the function of the fluorinases.

Metabolic Engineering of E. coli for Xylose Production from Glucose as the Sole Carbon Source.

Xylose is the raw material for the synthesis of many important platform compounds. At present, xylose is commercially produced by chemical extraction. However, there are still some bottlenecks in the extraction of xylose, including complicated operation processes and the chemical substances introduced, leading to the high cost of xylose and of synthesizing the downstream compounds of xylose. The current market price of xylose is 8× that of glucose, so using low-cost glucose as the substrate to produce the downstream compounds of xylose can theoretically reduce the cost by 70%. Here, we designed a pathway for the biosynthesis of xylose from glucose in Escherichia coli. This biosynthetic pathway was achieved by overexpressing five genes, namely, zwf, pgl, gnd, rpe, and xylA, while replacing the native xylulose kinase gene xylB with araL from B. subtilis, which displays phosphatase activity toward d-xylulose 5-phosphate. The yield of xylose was increased to 3.3 g/L by optimizing the metabolic pathway. Furthermore, xylitol was successfully synthesized by introducing the xyl1 gene, which suggested that the biosynthetic pathway of xylose from glucose is universally applicable for the synthesis of xylose downstream compounds. This is the first study to synthesize xylose and its downstream compounds by using glucose as a substrate, which not only reduces the cost of raw materials, but also alleviates carbon catabolite repression (CCR), providing a new idea for the synthesis of downstream compounds of xylose.

Study on the isoprene-producing co-culture system of Synechococcus elongates-Escherichia coli through omics analysis.

BACKGROUND: The majority of microbial fermentations are currently performed in the batch or fed-batch manner with the high process complexity and huge water consumption. The continuous microbial production can contribute to the green sustainable development of the fermentation industry. The co-culture systems of photo-autotrophic and heterotrophic species can play important roles in establishing the continuous fermentation mode for the bio-based chemicals production. RESULTS: In the present paper, the co-culture system of Synechococcus elongates-Escherichia coli was established and put into operation stably for isoprene production. Compared with the axenic culture, the fermentation period of time was extended from 100 to 400 h in the co-culture and the isoprene production was increased to eightfold. For in depth understanding this novel system, the differential omics profiles were analyzed. The responses of BL21(DE3) to S. elongatus PCC 7942 were triggered by the oxidative pressure through the Fenton reaction and all these changes were linked with one another at different spatial and temporal scales. The oxidative stress mitigation pathways might contribute to the long-lasting fermentation process. The performance of this co-culture system can be further improved according to the fundamental rules discovered by the omics analysis. CONCLUSIONS: The isoprene-producing co-culture system of S. elongates-E. coli was established and then analyzed by the omics methods. This study on the co-culture system of the model S. elongates-E. coli is of significance to reveal the common interactions between photo-autotrophic and heterotrophic species without natural symbiotic relation, which could provide the scientific basis for rational design of microbial community.

An Aminotransferase from Enhydrobacter aerosaccus to Obtain Optically Pure ß-Phenylalanine.

An aminotransferase ω-TAEn was identified from Enhydrobacter aerosaccus. The ω-TAEn was successfully expressed in Escherichia coli and the obtained enzyme showed activity toward ß-phenylalanine (ß-phe) at optimal conditions. For optically pure (R)-ß-phe, 50% yield was observed by kinetic resolution of racemic amino with pyruvate as the amino acceptor. To obtain (S)-ß-phe, the lipase/ω-TAEn catalytic system was adopted. The ω-TAEn showed strict stereoselectivity to the amino donor. The formation of (S)-ß-phe was observed using 3-aminobutyric acid as the amino donor, and (S)-ß-phe was obtained by asymmetric synthesis with a yield of 82%.

Increasing the pyruvate pool by overexpressing phosphoenolpyruvate carboxykinase or triosephosphate isomerase enhances phloroglucinol production in Escherichia coli.

OBJECTIVES: Acetyl-CoA is a precursor for phloroglucinol (PG), and pyruvate is one of the sources of intracellular acetyl-CoA. Therefore, enhancing intracellular pyruvate levels may help to improve the anabolic pathway of PG. RESULTS: In this study, the effects of phosphoenolpyruvate carboxykinase (PckA, encoded by pckA) or triosephosphate isomerase (TpiA, encoded by tpiA) overexpression on the production of PG were studied. Overexpression of pckA or tpiA could enhance the pyruvate anabolic pathway in shake-flask culture compared to the control strain, and the concentration of PG also increased by 44% and 92%, respectively. In addition, the acetate levels were all down regulated by the overexpression of the two genes to some extent and lower acetate level resulted in lower ATP pool and higher survival rate. CONCLUSIONS: These results indicate that overexpression of pckA or tpiA can enhance the pyruvate "pool" and PG production in Escherichia coli, which provides a new reference for further increasing the production of PG.

Metabolic engineering of Escherichia coli for the utilization of ethanol.

BACKGROUND: The fuel ethanol industry has made tremendous progress in the last decades. Ethanol can be obtained by fermentation using a variety of biomass materials as the feedstocks. However, few studies have been conducted on ethanol utilization by microorganisms. The price of petroleum-derived ethanol, easily made by the hydrolysis of ethylene, is even lower than that of bioethanol. If ethanol can be metabolized by microorganisms to produce value-added chemicals, it will open a new door for the utilization of inexpensive ethanol resources. RESULTS: We constructed an engineered Escherichia coli strain which could utilize ethanol as the sole carbon source. The alcohol dehydrogenase and aldehyde dehydrogenase from Aspergillus nidulans was introduced into E. coli and the recombinant strain acquired the ability to grow on ethanol. Cell growth continued when ethanol was supplied after glucose starvation and 2.24 g L-1 of ethanol was further consumed during the shake-flasks fermentation process. Then ethanol was further used for the production of mevalonic acid by heterologously expressing its biosynthetic pathway. Deuterium-labeled ethanol-D6 as the feedstock confirmed that mevalonic acid was synthesized from ethanol. CONCLUSIONS: This study demonstrated the possibility of using ethanol as the carbon source by engineered E. coli strains. It can serve as the basis for the construction of more robust strains in the future though the catabolic capacity of ethanol should be further improved.

Improving the production of isoprene and 1,3-propanediol by metabolically engineered Escherichia coli through recycling redox cofactor between the dual pathways.

The biosynthesis of isoprene by microorganisms is a promising green route. However, the yield of isoprene is limited due to the generation of excess NAD(P)H via the mevalonate (MVA) pathway, which converts more glucose into CO2 or undesired reduced by-products. The production of 1,3-propanediol (1,3-PDO) from glycerol is a typical NAD(P)H-consuming process, which restricts 1,3-PDO yield to ~ 0.7 mol/mol. In this study, we propose a strategy of redox cofactor balance by coupling the production of isoprene with 1,3-PDO fermentation. With the introduction and optimization of the dual pathways in an engineered Escherichia coli, ~ 85.2% of the excess NADPH from isoprene pathway was recycled for 1,3-PDO production. The best strain G05 simultaneously produced 665.2 mg/L isoprene and 2532.1 mg/L 1,3-PDO under flask fermentation conditions. The yields were 0.3 mol/mol glucose and 1.0 mol/mol glycerol, respectively, showing 3.3- and 4.3-fold improvements relative to either pathway independently. Since isoprene is a volatile organic compound (VOC) whereas 1,3-PDO is separated from the fermentation broth, their coproduction process does not increase the complexity or cost for the separation from each other. Hence, the presented strategy will be especially useful for developing efficient biocatalysts for other biofuels and biochemicals, which are driven by cofactor concentrations.

Electricigens in the anode of microbial fuel cells: pure cultures versus mixed communities.

Microbial fuel cell (MFC) is an environmentally friendly technology for electricity harvesting from a variety of substrates. Microorganisms used as catalysts in the anodic chamber, which are termed as electricigens, play a major role in the operation of MFCs. This review provides an introduction to the currently identified electricigens on their taxonomical groups and electricity producing abilities. The mechanism of electron transfer from electricigens to electrode is highlighted. The performances of pure culture and mixed communities are compared particularly. It has been proved that the electricity generation capacity and the ability to adapt to the complex environment of MFC systems constructed by pure microbial cultures are less than the systems constructed by miscellaneous consortia. However, pure cultures are useful to clarify the electron transfer mechanism at the microbiological level and further reduce the complexity of mixed communities. Future research trends of electricigens in MFCs should be focused on screening, domestication, modification and optimization of multi-strains to improve their electrochemical activities. Although the MFC techniques have been greatly advanced during the past few years, the present state of this technology still requires to be combined with other processes for cost reduction.

Manipulation of the precursor supply for high-level production of longifolene by metabolically engineered Escherichia coli.

Longifolene is a naturally occurring tricyclic sesquiterpene widely used in many different fields. Up to now, this valuable terpene was mainly manufactured from the high-boiling fraction of certain pine resins. Microbial production can be a promising alternative to the extraction from natural plant sources. Here, we present the metabolic engineering strategy to assemble biosynthetic pathway for longifolene production in Escherichia coli. E. coli was rendered to produce longifolene by heterologously expressing a codon optimized longifolene synthase from Picea abies. Augmentation of the metabolic flux to farnesyl pyrophosphate (FPP) by different FPP synthases conferred a 1.8-fold increase in longifolene production. An additional enhancement of longifolene production (up to 2.64 mg/L) was achieved by introducing an exogenous mevalonate pathway. Under fed-batch conditions, the best-performing strain was able to produce 382 mg/L of longifolene in a 5 L bioreactor. These results demonstrated the feasibility of producing longifolene by microbial fermentation and could serve as the basis for the construction of more robust strains in the future.

Biosynthesis and production of sabinene: current state and perspectives.

Sabinene is an important naturally occurring bicyclic monoterpene which can be used as flavorings, perfume additives, fine chemicals, and advanced biofuels. Up to now, this valuable terpene is commercially unavailable since there is no applicable manufacturing process. Microbial synthesis can be a promising route for sabinene production. In this review, we summarize knowledge about the metabolic pathway and key enzymes for sabinene biosynthesis. Recent advances that have been made in production of sabinene by microbial fermentation are highlighted. In these studies, researchers have identified the general synthetic pathway of sabinene from simple intermediate metabolites. Sabinene synthases of different origins were also cloned and characterized. Additionally, heterologous systems of the model microbes Escherichia coli and Saccharomyces cerevisiae were constructed to produce sabinene. This review also suggests new directions and attempts to gain some insights for achieving an industrial level production of sabinene. The combination of traditional molecular biology with new genome and proteome analysis tools will provide a better view of sabinene biosynthesis and a greater potential of microbial production.

Improving phloroglucinol tolerance and production in Escherichia coli by GroESL overexpression.

BACKGROUND: Phloroglucinol is an important chemical which has been successfully produced by engineered Escherichia coli. However, the toxicity of phloroglucinol can enormously inhibit E. coli cell growth and viability, and the productivity is still too low and not economically feasible for industrial applications. Therefore, strain tolerance to toxic metabolites remains a key issue during the production of chemicals using biological processes. RESULTS: In the present work, we examined the impact of the native GroESL chaperone system with different overexpression levels on phloroglucinol tolerance and production in E. coli. The groESL gene was cloned into an expression vector, of which expression level was regulated by three different promoters (natural, tac and T7 promoter). Strain tolerance was evaluated employing viable cell counts and phloroglucinol production. In comparison with the control strain, all GroESL overexpressing strains showed good characteristics in cell viability and phloroglucinol synthesis. Strain which overexpressed GroESL under tac promoter was found to show the best tolerance in all of those tested, resulting in a 3.19-fold increase in viable cell numbers compared with control strain of agar-plate culture under the condition of 0.7 g/L phloroglucinol, and a 39.5% increase in phloroglucinol production under fed-batch fermentation. This engineered strain finally accumulated phloroglucinol up to 5.3 g/L in the fed-batch cultivation 10 h after induction, and the productivity was 0.53 g/L/h. To date, the highest phloroglucinol production was achieved in this work compared with the previous reports, which is promising to make the bioprocess feasible from the economical point. CONCLUSIONS: The data show that appropriate expression level of GroESL plays a critical role in improving phloroglucinol tolerance and production in E. coli, and maybe involve in controlling some aspects of the stress response system through upregulation of GroESL. GroESL overexpression is therefore a feasible and efficient approach for improvement of E. coli tolerance.

An in vitro synthetic biosystem based on acetate for production of phloroglucinol.

BACKGROUND: Phloroglucinol is an important chemical, and the biosynthesis processes which can convert glucose to phloroglucinol have been established. However, due to approximate 80% of the glucose being transformed into undesirable by-products and biomass, this biosynthesis process only shows a low yield with the highest value of about 0.20 g/g. The industrial applications are usually hindered by the low current productivity and yield and also by the high costs. Generally, several different aspects limit the development of phloroglucinol biosynthesis. The yield of phloroglucinol is one of the most important parameters for its bioconversion especially from economic and ecological points of view. The in vitro biosynthesis of bio-based chemicals, is a flexible alternative with potentially high-yield to in vivo biosynthetic technology. RESULTS: By comparing the activity of acetyl-CoA synthetase (ACS) from Escherichia coli and Acetobacter pasteurianus, the highly active ACS2 was identified in A. pasteurianus. Acetyl-CoA carboxylase (ACC) from Acinetobacter calcoaceticus and phloroglucinol synthase (PhlD) from Pseudomonas fluorescens pf-5 were expressed and purified. Acetate was successfully transformed into phloroglucinol by the combined activity of above-mentioned enzymes and required cofactor. After optimization of the in vitro reaction system, phloroglucinol was then produced with a yield of nearly 0.64 g phloroglucinol/g acetic acid, which was equal to 91.43% of the theoretically possible maximum. CONCLUSIONS: In this work, a novel in vitro synthetic system for a highly efficient production of phloroglucinol from acetate was demonstrated. The system's performance suggests that in vitro synthesis of phloroglucinol has some advantages and is potential to become a feasible industrial alternative. Based on the results presented herewith, it is believed that in vitro biosystem will provide a feasible option for production of important industrial chemicals from acetate, which could work as a versatile biosynthetic platform.

Imidazolium-based ionic liquids for cellulose pretreatment: recent progresses and future perspectives.

As the most abundant biomass in nature, cellulose is considered to be an excellent feedstock to produce renewable fuels and fine chemicals. Due to its hydrogen-bonded supramolecular structure, cellulose is hardly soluble in water and most conventional organic solvents, limiting its further applications. The emergence of ionic liquids (ILs) provides an environmentally friendly, biodegradable solvent system to dissolve cellulose. This review summarizes recent advances concerning imidazolium-based ILs for cellulose pretreatment. The structure of cations and anions which has an influence on the solubility is emphasized. Methods to assist cellulose pretreatment with ILs are discussed. The state of art of the recovery, regeneration, and reuse aspects of ILs is also presented in this work. The current challenges and development directions of cellulose dissolution in ILs are put forward. Although further studies are still much required, commercialization of IL-based processes has made great progress in recent years.

High-specificity synthesis of novel monomers by remodeled alcohol hydroxylase.

BACKGROUND: Diols are important monomers for the production of plastics and polyurethanes, which are widely used in our daily life. The medium-chain diols with one hydroxyl group at its subterminal end are able to confer more flexibility upon the synthesized materials. But unfortunately, this type of diols has not been synthesized so far. The strong need for advanced materials impelled us to develop a new strategy for the production of these novel diols. In this study, we use the remodeled P450BM3 for high-specificity production of 1,7-decanediol. RESULTS: The native P450BM3 was capable of converting medium-chain alcohols into corresponding α, ω1-, α, ω2- and α, ω3-diols, with each of them accounting for about one third of the total diols, but it exhibited a little or no activity on the short-chain alcohols. Greatly improved regiospecificity of alcohol hydroxylation was obtained by laboratory evolution of P450BM3. After substitution of 12 amino acid residues (J2-F87A), the ratio of 1,7-decanediol (ω-3 hydroxylation) to total decanediols increased to 86.8 % from 34.0 %. Structure modeling and site-directed mutagenesis demonstrated that the heme end residues such as Ala(78), Phe(87) and Arg(255) play a key role in controlling the regioselectivity of the alcohol hydroxylation, while the residues at the mouth of substrate binding site is not responsible for the regioselectivity. CONCLUSIONS: Herein we employ an engineered P450BM3 for the first time to enable the high-specificity biosynthesis of 1,7-decanediol, which is a promising monomer for the development of advanced materials. Several key amino acid residues that control the regioselectivity of alcohol hydroxylation were identified, providing some new insights into how to improve the regiospecificity of alcohol hydroxylation. This report not only provides a good strategy for the biosynthesis of 1,7-decanediol, but also gives a promising approach for the production of other useful diols.