Progress in research on the biosynthesis of 1,2,4-butanetriol by engineered microbes.

1,2,4-butanetriol (BT) is a polyol with unique chemical properties, which has a stereocenter and can be divided into D-BT (the S-enantiomer) and L-BT (the R-enantiomer). BT can be used for the synthesis of 1,2,4-butanetriol trinitrate, 3-hydroxytetrahydrofuran, polyurethane, and other chemicals. It is widely used in the military industry, medicine, tobacco, polymer. At present, the BT is mainly synthesized by chemical methods, which are accompanied by harsh reaction conditions, poor selectivity, many by-products, and environmental pollution. Therefore, BT biosynthesis methods with the advantages of mild reaction conditions and green sustainability have become a current research hotspot. In this paper, the research status of microbial synthesis of BT was summarized from the following three aspects: (1) the biosynthetic pathway establishment for BT from xylose; (2) metabolic engineering strategies employed for improving BT production from xylose; (3) other substrates for BT production. Finally, the challenges and prospects of biosynthetic BT were discussed for future methods to improve competitiveness for industrial production.

Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol.

BACKGROUND: (R)-(+)-perillyl alcohol is a naturally oxygenated monoterpene widely used as the natural flavor additives, insecticides, jet fuels and anti-cancer therapies. It was also readily available monoterpene precursors. However, this natural product is present at low concentrations from plant sources which are not economically viable. Therefore, alternative microbial production methods are rapidly emerging as an attractive alternative to make (R)-(+)-perillyl alcohol production more sustainable and environmentally friendly. RESULTS: We engineered Escherichia coli to possess a heterologous mevalonate (MVA) pathway, including limonene synthase, P-cymene monoxygenase hydroxylase and P-cymene monoxygenase reductase for the production of (R)-(+)-perillyl alcohol. The concentration of (R)-(+)-limonene (the monoterpene precursor to (R)-(+)-perillyl alcohol) reached 45 mg/L from glucose. Enhanced (R)-(+)-perillyl alcohol production was therefore achieved. The strain produced (R)-(+)-perillyl alcohol at a titer of 87 mg/L and a yield of 1.5 mg/g glucose in a 5 L bioreactor fed batch system. CONCLUSIONS: These datas highlight the efficient production of (R)-(+)-perillyl alcohol through the mevalonate pathway from glucose. This method serves as a platform for the future production of other monoterpenes.

Recent advances in the biosynthesis of isoprenoids in engineered Saccharomyces cerevisiae.

Isoprenoids, as the largest group of chemicals in the domains of life, constitute more than 50,000 members. These compounds consist of different numbers of isoprene units (C5H8), by which they are typically classified into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30), and tetraterpenoids (C40). In recent years, isoprenoids have been employed as food additives, in the pharmaceutical industry, as advanced biofuels, and so on. To realize the sufficient and efficient production of valuable isoprenoids on an industrial scale, fermentation using engineered microorganisms is a promising strategy compared to traditional plant extraction and chemical synthesis. Due to the advantages of mature genetic manipulation, robustness and applicability to industrial bioprocesses, Saccharomyces cerevisiae has become an attractive microbial host for biochemical production, including that of various isoprenoids. In this review, we summarized the advances in the biosynthesis of isoprenoids in engineered S. cerevisiae over several decades, including synthetic pathway engineering, microbial host engineering, and central carbon pathway engineering. Furthermore, the challenges and corresponding strategies towards improving isoprenoid production in engineered S. cerevisiae were also summarized. Finally, suggestions and directions for isoprenoid production in engineered S. cerevisiae in the future are discussed.

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.

Biosynthesis of 2,4-diacetylphloroglucinol from glucose using engineered Escherichia coli.

In order to produce 2,4-diacetylphloroglucinol (2,4-DAPG) in E. coli, the key synthases coding by phlACBD gene cluster from the strain Pseudomonas fluorescens CHA0 were overexpressed in E. coli BL21 (DE3). The marA, phlE and acc genes were also overexpressed to enhance 2,4-DAPG biosynthesis. Then the fermentation conditions were optimized to improve the concentration of 2,4-DAPG. The results showed that the recombinant E. coli could produce few 2,4-DAPG with only the phlACBD gene cluster. The synthetic ability of 2,4-DAPG could be increased by expressing the acc, marA and phlE genes in shake-flasks cultivation. The effects of phloroglucinol, initial pH, temperature and trace elements on 2,4-DAPG biosynthesis were also investigated. Based on the optimal fermentation conditions obtained from the shake-flasks cultivation, fed-batch fermentation of strain Z3 in a 5 L bioreactor was conducted to produce 2,4-DAPG. Finally, the concentration of 2,4-DAPG was 179 mg/L after induction for 36 h by fed-batch fermentation. To the best of our knowledge, this is the highest 2,4-DAPG production reported in E. coli. This work showed the potential application of engineered E. coli to get high production of target compounds.

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.

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.

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.

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.

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.

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.

Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis.

3-Hydroxypropionate (3HP) is an important platform chemical, and four 3HP biosynthetic routes were reported, in which the malonyl-CoA pathway has some expected advantages but presented the lowest 3HP yield. Here, we demonstrated that this low yield was caused by a serious functional imbalance between MCR-C and MCR-N proteins, responsible for the two-step reduction of malonyl-CoA to 3HP. Then we minimized the enzyme activity imbalance by directed evolution of rate-limiting enzyme MCR-C and fine tuning of MCR-N expression level. Combined with culture conditions optimization, our engineering approaches increased the 3HP titer 270-fold, from 0.15 g/L to 40.6 g/L, representing the highest 3HP production via malonyl-CoA pathway so far. This study not only significantly improved the 3HP productivity of recombinant Escherichia coli strain, but also proved the importance of metabolic balance in a multistep biosynthetic pathway, which should be always considered in any metabolic engineering study.

Highly Transparent, Temperature-Resistant, and Flexible Polyimide Aerogels for Solar Energy Collection.

Advanced aerogel materials with low thermal conductivity and high transparency have shown great application prospects in the solar thermal energy conversion field. However, most aerogels do not meet these requirements due to their low optical transparency and poor mechanical properties. To tackle this problem, we have created versatile polyimide (PI) aerogel materials by adjusting the monomers to alter their molecular structure. These materials exhibit exceptional thermal insulation properties and high transparency, making them ideal for use in the construction of efficient solar collector devices. Incorporating 1,3,5-benzenetricarbonyl trichloride into PI aerogel results in high strength (>3 MPa) and excellent transmittance (>90%) over a broad range of wavelengths (500-2650 nm). The as-prepared PI aerogel solar collector (PIASC) also exhibits a low thermal conductivity (0.032 W/mK), a low density (0.1 g/cm3), and high porosity (90%). By changing the shape of the collector from a flat plate to a cylindrical ring, the heat collection efficiency and capacity are significantly improved, resulting in efficient heat collection. The circular ring collector has a maximum heat collection temperature of 236.8 °C. The PIASC, which is both flexible and highly transparent, is an ideal candidate for advanced optical elements and solar collectors.

Characterization of an efficient N-oxygenase from Saccharothrix sp. and its application in the synthesis of azomycin.

BACKGROUND: The nitro group constitutes a significant functional moiety within numerous valuable substances, such as nitroimidazoles, a class of antimicrobial drugs exhibiting broad spectrum activity. Conventional chemical methods for synthesizing nitro compounds suffer from harsh conditions, multiple steps, and environmental issues. Biocatalysis has emerged as a promising alternative to overcome these drawbacks, with certain enzymes capable of catalyzing nitro group formation gradually being discovered in nature. Nevertheless, the practical application is hindered by the restricted diversity and low catalytic activity exhibited by the reported nitrifying enzymes. RESULTS: A novel N-oxygenase SaRohS harboring higher catalytic capability of transformation 2-aminoimidazole to azomycin was characterized from Saccharothrix sp. Phylogenetic tree analysis revealed that SaRohS belongs to the heme-oxygenase-like diiron oxygenase (HDOs) family. SaRohS exhibited optimal activity at pH 5.5 and 25 â„ƒ, respectively. The enzyme maintained relatively stable activity within the pH range of 4.5 to 6.5 and the temperature range of 20 â„ƒ to 35 â„ƒ. Following sequence alignment and structural analysis, several promising amino acid residues were meticulously chosen for catalytic performance evaluation. Site-directed mutations showed that threonine 75 was essential for the catalytic activity. The dual mutant enzyme G95A/K115T exhibited the highest catalytic efficiency, which was approximately 5.8-fold higher than that of the wild-type and 22.3-fold higher than that of the reported N-oxygenase KaRohS from Kitasatospora azatica. The underlying catalytic mechanism was investigated through molecular docking and molecular dynamics. Finally, whole-cell biocatalysis was performed and 2-aminoimidazole could be effectively converted into azomycin with a reaction conversion rate of 42% within 14 h. CONCLUSIONS: An efficient N-oxygenase that catalyzes 2-aminoimidazole to azomycin was screened form Saccharothrix sp., its phylogenetics and enzymatic properties were analyzed. Through site-directed mutation, enhancements in catalytic competence were achieved, and the molecular basis underlying the enhanced enzymatic activity of the mutants was revealed via molecular docking and dynamic simulation. Furthermore, the application potential of this enzyme was assessed through whole cell biocatalysis, demonstrating it as a promising alternative method for azomycin production.

Cloning and transcription analysis of the Candida rugosa propionyl-CoA dehydrogenase gene and its expression in Pichia pastoris.

The propionyl-CoA dehydrogenase (PACD) gene was cloned from Candida rugosa by the cDNA RACE technique. The full cDNA of the PACD gene has a length of 1408 bp, which contains a complete open reading frame (ORF) of 1329 bp, coding for 442 amino acids. The cDNA of PACD was cloned into the expression plasmid pPIC9K and transformed into Pichia pastoris GS115. The recombinant protein was purified by Ni-NTA affinity chromatography, and its size was observed to be approximately 49 kDa as estimated by SDS-PAGE. Anti-His antibodies were used to characterise the recombinant PACD by western-blot analysis. The recombinant protein retained the activity of catalysing propionyl-CoA to acryloyl-CoA. The results of dot-blotting hybridisation using a PACD cDNA probe indicated that the PACD mRNA level was modified at different stages: mRNA levels were low for the first 36 h, then increased through 48 h and eventually reached a stable level. These results indicate that propionate induction could significantly activate PACD mRNA expression. Information from this study will be helpful in elucidating the metabolic pathway for 3-hydroxypropionic acid production in C. rugosa.

Biosynthesis of (R)-(+)-perillyl alcohol by Escherichia coli expressing neryl pyrophosphate synthase.

(R)-(+)-perillyl alcohol is widely used in agricultural and anticarcinogenic fields. Microbial production of (R)-(+)-perillyl alcohol was investigated in this study. We optimized biosynthesis of (R)-(+)-perillyl alcohol in Escherichia coli by using neryl pyrophosphate synthase and NADPH regeneration. Engineering neryl pyrophosphate (NPP)-supplied pathway resulted in a 4-fold improvement of (R)-(+)-perillyl alcohol titer. Subsequently, combined engineering of p-cymene monooxygenase (CymA) expression and module for NADPH regeneration exhibited a 15.4-fold increase of titer over the initial strain S02. Finally, 453 mg/L (R)-(+)-perillyl alcohol was achieved in fed-batch fermentation, which is the highest (R)-(+)-perillyl alcohol titer in E. coli.

Efficient Synthesis of (R)-(+)-Perillyl Alcohol From (R)-(+)-Limonene Using Engineered Escherichia coli Whole Cell Biocatalyst.

(R)-(+)-perillyl alcohol is a much valued supplemental compound with a wide range of agricultural and pharmacological characteristics. The aim of this study was to improve (R)-(+)-perillyl alcohol production using a whole-cell catalytic formula. In this study, we employed plasmids with varying copy numbers to identify an appropriate strain, strain 03. We demonstrated that low levels of alKL provided maximal biocatalyst stability. Upon determination of the optimal conditions, the (R)-(+)-perillyl alcohol yield reached 130 mg/L. For cofactor regeneration, we constructed strain 10, expressing FDH from Candida boidinii, and achieved (R)-(+)-perillyl alcohol production of 230 mg/L. As a result, 1.23 g/L (R)-(+)-perillyl alcohol was transformed in a 5 L fermenter. Our proposed method facilitates an alternative approach to the economical biosynthesis of (R)-(+)-perillyl alcohol.

Highly efficient biosynthesis of ß-caryophyllene with a new sesquiterpene synthase from tobacco.

BACKGROUND: ß-Caryophyllene, a kind of bicyclic sesquiterpene, is mainly used as a spice in the food and cosmetic industries. Furthermore, it also has significant value in the pharmaceutical industry and is now considered to be used as a new fuel. As a chemical energy heterotrophic microorganism, Escherichia coli can produce a large amount of acetyl-CoA through aerobic respiration, and acetyl-CoA is the common precursor substance in the biosynthesis of all terpenoids. Therefore, E. coli has the potential to be a cell factory to produce terpenoids. RESULTS: A new gene of ß-caryophyllene synthase (TPS7) was found by analyzing the genome of Nicotiana tabacum L. using bioinformatics methods. The gene was overexpressed in engineered E. coli with a heterogeneous mevalonate (MVA) pathway to build a recombinant strain CAR1. Subsequent cultivation experiments in shake flask of engineered strain CAR1 verified that 16.1 mg/L ß-caryophyllene was detected from the fermentation broth in the shake flask after induction for 24 h with IPTG. The toxic by-product of farnesyl acetate was detected during the process, and CAR1 showed a heavily cellular accumulation of product. We constructed an engineered strain CAR2, in which the downstream genes of the MVA pathway were integrated into the E. coli chromosome, successfully increasing ß-caryophyllene production to 100.3 mg/L. The highest production of ß-caryophyllene during the fed-batch fermentation was 4319 mg/L. Then we employed in situ extraction fermentation to successfully increase the production of ß-caryophyllene by 20% to 5142 mg/L. CONCLUSION: A new sesquiterpene synthase, TPS7, from tobacco was found to be able to produce ß-caryophyllene with high efficiency. Based on this, an engineered E. coli was constructed to produce a much higher concentration of ß-caryophyllene than the previous studies. During the fermentation process, we observed that ß-caryophyllene tends to accumulate in intracellular space, which will eventually influence the activity of engineered E. coli. As a result, we solved this by metabolism regulation and in situ extractive fermentation.

Improved phloroglucinol production by metabolically engineered Escherichia coli.

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.

Glycerol/glucose co-fermentation: one more proficient process to produce propionic acid by Propionibacterium acidipropionici.

Cosubstrates fermentation is such an effective strategy for increasing subject metabolic products that it could be available and studied in propionic acid production, using glycerol and glucose as carbon resources. The effects of glycerol, glucose, and their mixtures on the propionic acid production by Propionibacterium acidipropionici CGMCC1.2225 (ATCC4965) were studied, with the aim of improving the efficiency of propionic acid production. The propionic acid yield from substrate was improved from 0.475 and 0.303 g g(-1) with glycerol and glucose alone, respectively, to 0.572 g g(-1) with co-fermentation of a glycerol/glucose mixture of 4/1 (mol/mol). The maximal propionic acid and substrate conversion rate were 21.9 g l(-1) and 57.2% (w/w), respectively, both significantly higher than for a sole carbon source. Under optimized conditions of fed-batch fermentation, the maximal propionic acid yield and substrate conversion efficiency were 29.2 g l(-1) and 54.4% (w/w), respectively. These results showed that glycerol/glucose co-fermentation could serve as an excellent alternative to conventional propionic acid fermentation.