Four-carbon Dicarboxylic Acid Research Articles

Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol.

The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in Yarrowia lipolytica, an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain Po1g-CEE2+Sp to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.

Reprogramming protein stability in Escherichia coli to improve four-carbon dicarboxylic acids production

Microbial cell factories (MCF) offer an eco-friendliness and cost-effectiveness way for producing high-value chemicals. However, their productive performance is often limited due to the loss of protein stability during microbial fermentation process. Here, protein stability was rationally reprogrammed to build efficient MCF for four-carbon dicarboxylic acids production by developing a codon combination subset-based protein stability regulator (ccsPSR). First, the effect of protein stability on fermentation production was analyzed, and then the targeted genes such as ribF responsible for protein stability were identified by transcriptomic analysis. Next, ccsPSR was designed and constructed by screening and analyzing synonymous codons capable of increasing adenine frequency from 2 to 6 codon positions in the ribF gene. Finally, by ccsPSR to adjust protein stability in E. coli, succinate production was up to 153.36 g/L with its productivity 2.13 g/L/h, and malate production was up to 30.02 g/L with its yield 1.04 g/g, respectively. These results suggest that reprogramming protein stability is an effective strategy for improving the production performance of MCF.

Metabolic and Microbial Community Engineering for Four-Carbon Dicarboxylic Acid Production from CO2-Derived Glycogen in the Cyanobacterium Synechocystis sp. PCC6803.

The four-carbon (C4) dicarboxylic acids, fumarate, malate, and succinate, are the most valuable targets that must be exploited for CO2-based chemical production in the move to a sustainable low-carbon future. Cyanobacteria excrete high amounts of C4 dicarboxylic acids through glycogen fermentation in a dark anoxic environment. The enhancement of metabolic flux in the reductive TCA branch in the Cyanobacterium Synechocystis sp. PCC6803 is a key issue in the C4 dicarboxylic acid production. To improve metabolic flux through the anaplerotic pathway, we have created the recombinant strain PCCK, which expresses foreign ATP-forming phosphoenolpyruvate carboxykinase (PEPck) concurrent with intrinsic phosphoenolpyruvate carboxylase (Ppc) overexpression. Expression of PEPck concurrent with Ppc led to an increase in C4 dicarboxylic acids by autofermentation. Metabolome analysis revealed that PEPck contributed to an increase in carbon flux from hexose and pentose phosphates into the TCA reductive branch. To enhance the metabolic flux in the reductive TCA branch, we examined the effect of corn-steep liquor (CSL) as a nutritional supplement on C4 dicarboxylic acid production. Surprisingly, the addition of sterilized CSL enhanced the malate production in the PCCK strain. Thereafter, the malate and fumarate excreted by the PCCK strain are converted into succinate by the CSL-settling microorganisms. Finally, high-density cultivation of cells lacking the acetate kinase gene showed the highest production of malate and fumarate (3.2 and 2.4 g/L with sterilized CSL) and succinate (5.7 g/L with non-sterile CSL) after 72 h cultivation. The present microbial community engineering is useful for succinate production by one-pot fermentation under dark anoxic conditions.

In vitro Enzyme Inhibition and In vivo Anti-hyperuricemic Potential of Malic Acid: An Experimental Approach

The prevalence of hyperuricemia among adolescents in developed and developing countries is of great concern. Hyperuricemia is a metabolic disorder that intensifies the risk of serious illnesses such as gout, diabetes, cardiovascular, renal complications etc. This can be controlled through regulating the xanthine oxidase (XO) enzyme activity. Current XO inhibitors used for the treatment exert some severe side effects. Naturally derived molecules are better alternative to overcome these issues. Malic acid, a four-carbon dicarboxylic acid, is mostly present in plants and fruits and is utilised extensively in the diet, chemical, and pharmaceutical industries. The IC50 value obtained for malic acid was 3.81 ± 0.002 × 10 M and it is a good competitive inhibitor. An altered levels of various biological markers (renal, hepatic and haematological) were observed in potassium oxonate induced in vivo hyperuricemia model. When treated with malic acid (different doses of 25, 50 and 100 mg/kg bwt), altered levels of biological function markers retained close to their normal range. The current study revealed the anti-hyperuricemic potential of malic acid.

Microbial Biosynthesis of L-Malic Acid and Related Metabolic Engineering Strategies: Advances and Prospects.

Malic acid, a four-carbon dicarboxylic acid, is widely used in the food, chemical and medical industries. As an intermediate of the TCA cycle, malic acid is one of the most promising building block chemicals that can be produced from renewable sources. To date, chemical synthesis or enzymatic conversion of petrochemical feedstocks are still the dominant mode for malic acid production. However, with increasing concerns surrounding environmental issues in recent years, microbial fermentation for the production of L-malic acid was extensively explored as an eco-friendly production process. The rapid development of genetic engineering has resulted in some promising strains suitable for large-scale bio-based production of malic acid. This review offers a comprehensive overview of the most recent developments, including a spectrum of wild-type, mutant, laboratory-evolved and metabolically engineered microorganisms for malic acid production. The technological progress in the fermentative production of malic acid is presented. Metabolic engineering strategies for malic acid production in various microorganisms are particularly reviewed. Biosynthetic pathways, transport of malic acid, elimination of byproducts and enhancement of metabolic fluxes are discussed and compared as strategies for improving malic acid production, thus providing insights into the current state of malic acid production, as well as further research directions for more efficient and economical microbial malic acid production.

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO2 using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO2 to carboxylic acids.

Extending the shikimate pathway for microbial production of maleate from glycerol in engineered Escherichia coli

Maleate is one of the most important unsaturated four-carbon dicarboxylic acids. It serves as an attractive building block in cosmetic, polymer, and pharmaceutical industries. Currently, industrial production of maleate relies mainly on chemical synthesis using benzene or butane as the starting materials under high temperature, which suffers from strict reaction conditions and low product yield. Here, we propose a novel biosynthetic pathway for maleate production in engineered Escherichia coli. We screened a superior salicylate 5-hydroxylase that can catalyze hydroxylation of salicylate into gentisate with high conversion rate. Then, introduction of salicylate biosynthetic pathway and gentisate ring cleavage pathway allowed the synthesis of maleate from glycerol. Further optimizations including enhancement of precursors supply, disruption of competing pathways, and construction of a pyruvate recycling system, boosted maleate titer to 2.4 ± 0.1 g/L in shake flask experiments. Subsequent scale-up biosynthesis of maleate in a 3-L bioreactor under fed-batch culture conditions enabled the production of 14.5 g/L of maleate, indicating a 268-fold improvement compared with the titer generated by the wildtype E. coli strain carrying the entire maleate biosynthetic pathway. This study provided a promising microbial platform for industrial level synthesis of maleate, and demonstrated the highest titer of maleate production in microorganisms so far.

Development of a Cell-recycled Continuous Fermentation Process for Enhanced Production of Succinic Acid by High-yielding Mutants of Actinobacillus succinogenes

Succinic acid (SA), a four-carbon dicarboxylic acid utilized as a platform chemical for valuable industrial products, is a major fermentation product of Actinobacillus succinogenes cells, synthetized during anaerobic metabolism. In this study, cell-recycled continuous fermentation (CRCF) was carried out in order to maximize the appreciably stable biocatalytic activity of SA-producing cells and the growth-associated mode of SA biosynthesis. Stable and long operations of CRCF could be carried out through an efficient decanter system developed in our laboratory, which could effectively separate highly dense microbial cells from the outlet stream. This allowed to overcome the wash-out phenomenon encountered at relatively low dilution rates in conventional continuous fermentation systems without cell recycling. Through careful assessment of the effects of dilution rate, composition of feeding medium, and cell recycling ratio on SA yield via the CRCF process, volumetric SA productivity could be enhanced to 3.86 g/(L·h), an approximately 5.1-fold increase compared to parallel continuous fermentation without cell recycling. A higher dilution rate and a 24% increment in SA production via increased density of active cells inside the bioreactor of the CRCF system were the probable factors inducing such a considerable enhancement in volumetric SA productivity during CRCF. Since volumetric productivity is the most important parameter determining the cost-effectiveness of a given fermentation bioprocess, it is quite evident that CRCF is a promising alternative to conventional batch or continuous fermentation without cell recycling for mass production of SA.

Optimization of anaerobic fermentation of Actinobacillus succinogenes for increase the succinic acid production

Succinic acid is a four-carbon dicarboxylic acid used as a building-block in the synthesis of valuable commodities and specialty chemicals. Succinic acid is produced petrochemically by hydrogenation of maleic anhydride and a “greener” alternative route is microbial fermentation. In this study, a 25−1 fractional factorial design (FFD) and a subsequent central composite rotatable design (CCRD) were used to investigate the influence of process parameters on succinic acid and biomass concentration during fermentation by Actinobacillus succinogenes. The independent factors were glucose concentration (19.29–42.45 g/L), yeast extract concentration (5–11 g/L), temperature (33–41 °C), pH (6–8), and agitation speed (100–300 rpm). Results showed that the main factors that influenced the succinic acid production were pH, yeast extract concentration and temperature. According to the CCRD results, the optimum conditions for succinic acid production were 37–41 °C, 13 g/L yeast extract concentration and pH 7. The maximum succinic acid concentration obtained was 17.5 g/L.

Recent progress on bio-based production of dicarboxylic acids in yeast.

Dicarboxylic acids are widely used in fine chemical and food industries as well as the monomer for polymerisation of high molecular material. Given the problems of environmental contamination and sustainable development faced by traditional production of dicarboxylic acids based on petrol, new approaches such as bio-based production of dicarboxylic acids drew more attentions. The yeast, Saccharomyces cerevisiae, was regarded as an ideal organism for bio-based production of dicarboxylic acids with high tolerance to acidic and hyperosmotic environments, robust growth using a broad range of substrates, great convenience for genetic manipulation, stable inheritance via sub-cultivation, and food compatibility. In this review, the production of major dicarboxylates via S. cerevisiae was concluded and the challenges and opportunities facing were discussed.Key Points• Summary of current production of major dicarboxylic acids by Saccharomyces cerevisiae.• Discussion of influence factors on four-carbon dicarboxylic acids production by Saccharomyces cerevisiae.• Outlook of potential production of five- and six-carbon dicarboxylic acids by Saccharomyces cerevisiae.

Fumarate production with Rhizopus oryzae: utilising the Crabtree effect to minimise ethanol by-product formation

BackgroundThe four-carbon dicarboxylic acids of the tricarboxylic acid cycle (malate, fumarate and succinate) remain promising bio-based alternatives to various precursor chemicals derived from fossil-based feed stocks. The double carbon bond in fumarate, in addition to the two terminal carboxylic groups, opens up an array of downstream reaction possibilities, where replacement options for petrochemical derived maleic anhydride are worth mentioning. To date the most promising organism for producing fumarate is Rhizopus oryzae (ATCC 20344, also referred to as Rhizopus delemar) that naturally excretes fumarate under nitrogen-limited conditions. Fumarate excretion in R. oryzae is always associated with the co-excretion of ethanol, an unwanted metabolic product from the fermentation. Attempts to eliminate ethanol production classically focus on enhanced oxygen availability within the mycelium matrix. In this study our immobilised R. oryzae process was employed to investigate and utilise the Crabtree characteristics of the organism in order to establish the limits of ethanol by-product formation under growth and non-growth conditions.ResultsAll fermentations were performed with either nitrogen excess (growth phase) or nitrogen limitation (production phase) where medium replacements were done between the growth and the production phase. Initial experiments employed excess glucose for both growth and production, while the oxygen partial pressure was varied between a dissolved oxygen of 18.4% and 85%. Ethanol was formed during both growth and production phases and the oxygen partial pressure had zero influence on the response. Results clearly indicated that possible anaerobic zones within the mycelium were not responsible for ethanol formation, hinting that ethanol is formed under fully aerobic conditions as a metabolic overflow product. For Crabtree-positive organisms like Saccharomyces cerevisiae ethanol overflow is manipulated by controlling the glucose input to the fermentation. The same strategy was employed for R. oryzae for both growth and production fermentations. It was shown that all ethanol can be eliminated during growth for a glucose addition rate of 0.07,hbox {g},hbox {L}^{-1},hbox {h}^{-1}. The production phase behaved in a similar manner, where glucose addition of 0.197,hbox {g},hbox {L}^{-1},hbox {h}^{-1} resulted in fumarate production of 0.150,hbox {g},hbox {L}^{-1},hbox {h}^{-1} and a yield of 0.802,hbox {g},hbox {g}^{-1} fumarate on glucose. Further investigation into the effect of glucose addition revealed that ethanol overflow commences at a glucose addition rate of 0.395,hbox {g},hbox {g}^{-1},hbox {h}^{-1} on biomass, while the maximum glucose uptake rate was established to be between 0.426 and 0.533,hbox {g},hbox {g}^{-1},hbox {h}^{-1}.ConclusionsThe results conclusively prove that R. oryzae is a Crabtree-positive organism and that the characteristic can be utilised to completely discard ethanol by-product formation. A state referred to as “homofumarate production” was illustrated, where all carbon input exits the cell as either fumarate or respiratory hbox {CO}_{2}. The highest biomass-based “homofumarate production”: rate of 0.243,hbox {g},hbox {g}^{-1},hbox {h}^{-1} achieved a yield of 0.802,hbox {g},hbox {g}^{-1} on glucose, indicating the bounds for developing an ethanol free process. The control strategy employed in this study in conjunction with the uncomplicated scalability of the immobilised process provides new direction for further developing bio-fumarate production.

Inactivation of Malic Enzymes Improves the Anaerobic Production of Four-Carbon Dicarboxylic Acids by Recombinant Escherichia coli Strains Expressing Pyruvate Carboxylase

The genes maeA and maeB, encoding NADH- and NADPH-dependent malic enzymes, have been deleted in a recombinant Escherichia coli strain with inactivated mixed-acid fermentation pathways and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. During anaerobic glucose utilization, the parental strain synthesized malic, fumaric, and succinic acids as the main fermentation end products, while pyruvic acid was accumulated as the main by-product resulting from the functioning of the pyruvate–oxaloacetate–malate–pyruvate futile cycle. Upon individual deletions of the maeA and maeB genes, the mutant strains converted glucose into four-carbon dicarboxylic acids with increased efficiency still secreting notable amounts of pyruvic acid. The combined inactivation of both malic enzymes in the constructed strain significantly elevated the portion of malic, fumaric, and succinic acids among the fermentation end products with a concomitant decrease in the secretion of pyruvic acid and other by-products due to the abolishment of the action of the futile cycle competing with the target biosynthetic processes.

High yield production of four-carbon dicarboxylic acids by metabolically engineered Escherichia coli.

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61-0.67mol (mole glucose)-1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38mM (5.41g/L) and 50.34mM (6.75g/L) malate with a molar yield of 0.53 and 0.55mol (mole succinate)-1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133mM (17.83g/L) malate in 47h, with a high yield of 1.3mol (mole glucose)-1 and productivity of 0.38gL-1h-1.

Инактивация малатных ферментов улучшает анаэробную продукцию четырехуглеродных дикарбоновых кислот рекомбинантными штаммами Escherichia coli, экспрессирующими пируваткарбоксилазу

Инактивация малатных ферментов улучшает анаэробную продукцию четырехуглеродных дикарбоновых кислот рекомбинантными штаммами Escherichia coli, экспрессирующими пируваткарбоксилазу

Metabolic engineering of Escherichia coli W3110 to produce L-malate.

A four-carbon dicarboxylic acid L-malate has recently attracted attention due to its potential applications in the fields of medicine and agriculture. In this study, Escherichia coli W3110 was engineered and optimized for L-malate production via one-step L-malate synthesis pathway. First, deletion of the genes encoding lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), pyruvate formate lyase (pflB), phosphotransacetylase (pta), and acetate kinase A (ackA) in pta-ackA pathway led to accumulate 20.9 g/L pyruvate. Then, overexpression of NADP+ -dependent malic enzyme C490S mutant in this multi-deletion mutant resulted in the direct conversion of pyruvate into L-malate (3.62 g/L). Next, deletion of the genes responsible for succinate biosynthesis further enhanced L-malate production up to 7.78 g/L. Finally, L-malate production was elevated to 21.65 g/L with the L-malate yield to 0.36 g/g in a 5 L bioreactor by overexpressing the pos5 gene encoding NADH kinase in the engineered E. coli F0931 strain. This study demonstrates the potential utility of one-step pathway for efficient L-malate production. Biotechnol. Bioeng. 2017;114: 656-664. © 2016 Wiley Periodicals, Inc.

The Roles of Organic Acids in C4 Photosynthesis.

Organic acids are involved in numerous metabolic pathways in all plants. The finding that some plants, known as C4 plants, have four-carbon dicarboxylic acids as the first product of carbon fixation showed these organic acids play essential roles as photosynthetic intermediates. Oxaloacetate (OAA), malate, and aspartate (Asp) are substrates for the C4 acid cycle that underpins the CO2 concentrating mechanism of C4 photosynthesis. In this cycle, OAA is the immediate, short-lived, product of the initial CO2 fixation step in C4 leaf mesophyll cells. The malate and Asp, resulting from the rapid conversion of OAA, are the organic acids delivered to the sites of carbon reduction in the bundle-sheath cells of the leaf, where they are decarboxylated, with the released CO2 used to make carbohydrates. The three-carbon organic acids resulting from the decarboxylation reactions are returned to the mesophyll cells where they are used to regenerate the CO2 acceptor pool. NADP-malic enzyme-type, NAD-malic enzyme-type, and phosphoenolpyruvate carboxykinase-type C4 plants were identified, based on the most abundant decarboxylating enzyme in the leaf tissue. The genes encoding these C4 pathway-associated decarboxylases were co-opted from ancestral C3 plant genes during the evolution of C4 photosynthesis. Malate was recognized as the major organic acid transferred in NADP-malic enzyme-type C4 species, while Asp fills this role in NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type plants. However, accumulating evidence indicates that many C4 plants use a combination of organic acids and decarboxylases during CO2 fixation, and the C4-type categories are not rigid. The ability to transfer multiple organic acid species and utilize different decarboxylases has been suggested to give C4 plants advantages in changing and stressful environments, as well as during development, by facilitating the balance of energy between the two cell types involved in the C4 pathway of CO2 assimilation. The results of recent empirical and modeling studies support this suggestion and indicate that a combination of transferred organic acids and decarboxylases is beneficial to C4 plants in different light environments.

Hydrothermal Conversion of Catechol into Four-Carbon Dicarboxylic Acids

Conversion of lignin into value-added chemicals is attracting growing attention due to the depletion of fossil fuels and the abundant resource of lignin. In this study, hydrothermal conversion of a model compound of lignin, catechol, into value-added four-carbon dicarboxylic acids (C4-DCAs), such as tartaric (HOOC–CH(OH)–CH(OH)–COOH), malic (HOOC–CH2–CH(OH)–COOH), and fumaric (HOOC—CH═CH—COOH) acids was investigated. The yield of total C4-DCAs can reach as high as 41.0%, and alkali played a key role in not only promoting the production but also avoiding the decomposition of C4-DCAs. The reaction mechanism of hydrothermal conversion catechol into C4-DCAs showed that catechol is first oxidized to o-quinone, which is then attacked by the hydroxyl radical (OH•) or the hydroperoxyl anion (HO2–) via conjugate addition to decompose into C4-DCAs. This result is helpful to facilitate studies for developing a new, green, and sustainable process to produce value-added C4-DCAs from lignin.

Actinobacillus succinogenes의 혐기성배양에 의해 생합성 되는 숙신산의 생산성 향상을 위한 통계적 생산배지 최적화

Statistical medium optimization has been carried out for the production of succinic acid in anaerobic fermentations of Actinobacillus succinogenes. Succinic acid utilized as a precursor of many industrially important chemicals is a fourcarbon dicarboxylic acid, biosynthesized as one of the fermentation products of anaerobic metabolism by A. succinogenes. Through OFAT (one factor at a time) experiments, corn steep liquor (CSL), a very cheap agricultural byproduct, was found to have significant effects on enhanced production of succinic acid, when supplemented along with yeast extract. Hence, using these factors including glucose as a carbon/energy source, interactive effects were investigated through <TEX>$2^n$</TEX> full factorial design (FFD) experiments, showing that the concentration of each component (i.e., glucose, yeast extract and CSL) should be higher. Further statistical experiments were conducted along the steepest ascent path, followed by response surface method (RSM) in order to find out optimal concentrations of each constituent. Consequently, optimized concentrations of glucose, yeast extract and CSL were observed to be 180 g/L, 15.08 g/L and 20.75 g/L respectively (10 g/L of <TEX>$NaHCO_3$</TEX> and 100 g/L of <TEX>$MgCO_3$</TEX> to be supplemented as bicarbonate suppliers), with the estimated production level of succinic acid to be 92.9 g/L (about 3.5 fold higher productivity as compared to the initial medium). Notably, the RSM-estimated production level was almost similar to the amount of succinic acid (92.9 g/L vs. 89.1 g/L) produced through the actual fermentation process performed using the statistically optimized production medium.

Succinate production in Escherichia coli

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.

High-throughput screening of high-yield colonies of Rhizopus oryzae for enhanced production of fumaric acid

Fumaric acid is an important four-carbon dicarboxylic acid as a potential biorefinery target. A high-throughput screening method for fumaric acid overproduction strains was established. Nystatin (50 mg/L) was added into the production medium to restrict the spread of Rhizopus oryzae hyphae on agar plates. With bromocerol green as a pH indicator in the agar plates, the capability of fumaric acid biosynthesis by single colony was positively correlated with the diameter ratio of the colored ring and the colony. With this novel strategy, one high-yield mutant (Rhizopus oryzae ZJU11) was isolated from a large colony library of Rhizopus oryzae after UV irradiation. Starting with an optimized glucose concentration of 85 g/L, Rhizopus oryzae ZJU11 can produce 57.4 g/L fumaric acid in flask and 41.1 g/L in 5-L fermentor, which were 205% and 160% higher than that of the parent strain, respectively. Further studies showed that the production of fumaric acid by Rhizopus oryzae ZJU11 remained at the same level after three consecutive generations on the fermentation medium.