Strain For Industrial Production Research Articles

Comparative genomics and transcriptomics analyses provide insights into the high yield and regulatory mechanism of Norvancomycin biosynthesis in Amycolatopsis orientalis NCPC 2-48

BackgroundNorvancomycin has been widely used in clinic to treat against MRSA (Methicillin-resistant Staphylococcus aureus) and MRSE (Methicillin-resistant Staphylococcus epidermidis) infections in China. Amycolatopsis orientalis NCPC 2-48, a high yield strain derived from A. orientalis CPCC 200066, has been applied in industrial large-scale production of norvancomycin by North China Pharmaceutical Group. However, the potential high-yield and regulatory mechanism involved in norvancomycin biosynthetic pathway has not yet been addressed.ResultsHere we sequenced and compared the genomes and transcriptomes of A. orientalis CPCC 200066 and NCPC 2-48. These two genomes are extremely similar with an identity of more than 99.9%, and no duplication and structural variation was found in the norvancomycin biosynthetic gene cluster. Comparative transcriptomic analysis indicated that biosynthetic genes of norvancomycin, as well as some primary metabolite pathways for the biosynthetic precursors of norvancomycin were generally upregulated. AoStrR1 and AoLuxR1, two cluster-situated regulatory genes in norvancomycin cluster, were 23.3-fold and 5.8-fold upregulated in the high yield strain at 48 h, respectively. Over-expression of AoStrR1 and AoLuxR1 in CPCC 200066 resulted in an increase of norvancomycin production, indicating their positive roles in norvancomycin biosynthesis. Furthermore, AoStrR1 can regulate the production of norvancomycin by directly interacting with at least 8 promoters of norvancomycin biosynthetic genes or operons.ConclusionOur results suggested that the high yield of NCPC 2-48 can be ascribed to increased expression level of norvancomycin biosynthetic genes in its cluster as well as the genes responsible for the supply of its precursors. The norvancomycin biosynthetic genes are presumably regulated by AoStrR1 and AoLuxR1, of them AoStrR1 is possibly the ultimate pathway-specific regulator for the norvancomycin production. These results are helpful for further clarification of the holistic and pathway-specific regulatory mechanism of norvancomycin biosynthesis in the industrial production strain.

Improving multiple stress-tolerance of a flocculating industrial Saccharomyces cerevisiae strain by random mutagenesis and hybridization

Breeding stress-tolerant industrial Saccharomyces cerevisiae strains is crucial to reduce the cost of bioethanol production. In this study, a novel strategy, atmospheric and room temperature plasma (ARTP) mutagenesis combined with genome shuffling and hybridization, was successfully applied to obtain a multiple stress-tolerant flocculating industrial S. cerevisiae strain E-158. The enhanced strain E-158 grew better under various stress conditions compared with the original strain KF-7. The concentrations of ethanol produced by strain E-158 during batch fermentation were 10.14 %–81.02 % higher than those produced by strain KF-7 under high ethanol, high temperature, and high osmosis stress conditions. The stress tolerance of E-158 kept stable even after 30 times of sub-cultivation. The lower reactive oxygen species (ROS) level and higher superoxide dismutase (SOD) and catalase (CAT) activities in strain E-158 manifested that the antioxidant defense ability of E-158 was improved and contributed to its multiple stress-tolerance. This study provided a stress-tolerant strain for industrial ethanol production and a feasible strategy for breeding multiple stress-tolerant industrial S. cerevisiae strain.

Identification and Characterization of an Efficient Phenylalanine Ammonia-Lyase from Photorhabdus luminescens.

A putative aromatic amino acid ammonia-lyase gene (named Pl-pal) was discovered in Photorhabdus luminescens DSM 3368. BLAST and phylogenetic analyses predicted that this enzyme is a histidine ammonia-lyase, whereas sequence alignment suggested that it is more likely a phenylalanine ammonia-lyase (PAL). This gene was amplified from P. luminescens and expressed in Escherichia coli BL21(DE3). The function of Pl-PAL (58 kDa) was characterized by in vitro enzymatic reactions with L-phenylalanine (L-Phe), L-tyrosine (L-Tyr), L-histidine (L-His), and L-tryptophan (L-Trp). Pl-PAL can convert L-Phe and L-Tyr to trans-cinnamic acid and p-coumaric acid, respectively, but had no function on L-His and L-Trp. The optimum temperature and pH were determined to be 40 °C and 11.0, respectively. Under the optimal conditions, Pl-PAL had a kcat/Km value of 0.52 s-1 mM-1 with L-Phe as the substrate, while only 0.013 s-1 mM-1 for L-Tyr. Therefore, the primary function of Pl-PAL was determined to be PAL. The Pl-pal-harboring E. coli strain was used as a whole-cell biocatalyst to produce trans-cinnamic acid from L-Phe. The overall molar conversion rate and productivity were 65.98% and 228.10 mg L-1 h-1, respectively, after the cells were repeatedly utilized 7 times. This work thus provides a promising strain for industrial production of trans-cinnamic acid.

Research advances on arachidonic acid production by fermentation and genetic modification of Mortierella alpina.

Arachidonic acid (ARA, 5, 8, 11, 14-cis-eicosatetraenoic acid) is a relevant ω-6 polyunsaturated fatty acid, which plays essential roles in human immune, cardiovascular, and nervous systems. It is widely used in medicine, cosmetics, nutrition, and other fields. Traditionally, ARA is obtained from animal tissues. However, due to the limitation and unsustainability of existing resources, microorganisms are a potential alternative resource for ARA production. In this regard, major efforts have been made on algae and filamentous fungi, among which Mortierella alpina is the most effective strain for industrial ARA production. In this review, we summarized the recent progress in enhancing M. alpina production by optimization of culture medium and fermentation process and genetic modification. In addition, we provided perspectives in synthetic biology methods and technologies to further increase ARA production.

Aerobic Utilization of Methanol for Microbial Growth and Production.

Methanol is a reduced one-carbon (C1) compound. It supports growth of aerobic methylotrophs that gain ATP from reduced redox equivalents by respiratory phosphorylation in their electron transport chains. Notably, linear oxidation of methanol to carbon dioxide may yield three reduced redox equivalents if methanol oxidation is NAD-dependent as, e.g., in Bacillus methanolicus. Methanol has a higher degree of reduction per carbon than glucose (6 vs. 4), and thus, lends itself as an ideal carbon source for microbial production of reduced target compounds. However, C-C bond formation in the RuMP or serine cycle, a prerequisite for production of larger molecules, requires ATP and/or reduced redox equivalents. Moreover, heat dissipation and a high demand for oxygen during catabolic oxidation of methanol may pose challenges for fermentation processes. In this chapter, we summarize metabolic pathways for aerobic methanol utilization, aerobic methylotrophs as industrial production hosts, strain engineering, and methanol bioreactor processes. In addition, we provide technological and market outlooks.

English

The performance of a hybrid of Saccharomyces cerevisiae and Saccharomyces paradoxus (Yeast B) compared with an industrial strain of S. cerevisiae (Yeast A) was studied. The two strains of yeasts: Yeast A obtained from the Bioresources Development Centre, National Biotechnology Development Agency Ogbomosho and Yeast B obtained from Scotch Whisky Research Laboratory in Edinburgh, Scotland (Strain 63M) were studied using the SLSF method for bioethanol production at various initial starch concentrations (20, 30 and 50%). The cassava starch used was extracted from Tropical Manihot esculenta (TME 419) cassava strain while the SLSF method for bioethanol production was initiated by adding granular starch hydrolysing enzyme with the yeast strains into the starch solutions at room temperature. The results obtained showed that Yeast B has higher bioethanol yield and tolerance generating 70.34, 87.34 and 120.53 ml/L compared to the Yeast A which produced 63.4, 72.73 and 112.6 ml/L at 20, 30 and 50% starch concentrations respectively. The study suggests that the hybrid strain of S. cerevisiae and S. paradoxus out performs the industrial strain and can favorably substitute or displace the industrial strains for bioethanol production. Key words: Bioethanol, cassava, fermentation, hybrid, simultaneous liquefaction saccharification and fermentation (SLSF), yeast, Saccharomyces cerevisiae, Saccharomyces paradoxus.

Hybrid genome assembly and functional annotation reveals insights on lipid biosynthesis of oleaginous native isolate Parachlorella kessleri, a potential industrial strain for production of biofuel precursors

The need for mass production of biofuels has been pushed up by rising energy demand in the world population. In this context, microalgae have emerged as a potential source for biodiesel production because of its high lipid content and ability to grow on minimal media sources. However, the knowledge of the metabolic processes in these systems is fairly naive and therefore hinders the genetic progress that can be proven to improve the development of biofuel precursors. In the present study, a comprehensive analysis of an indigenous oleaginous microalgae P. kessleri has been performed, using hybrid assembly of both short Illumina and long Nanopore read, leading to assembly of a highly contiguous 64.90 Mb genome with 8371 functional genes and 874 putative transcription factors. The genome reconstruction is complemented using phylogenetic analysis of acyl lipid metabolism revealed the evolutionary conservation of these proteins among various microalgal species. Henceforth, understanding and reconstruction of global metabolic pathways will provide clues towards engineering of algal strains for enhanced production of biomass, biofuels and bioproducts (B3).

Biotechnological Advances in Lycopene β-Cyclases.

Lycopene β-cyclase is one of the key enzymes in the biosynthesis of carotenoids, which catalyzes the β-cyclization of both ends of lycopene to produce β-carotene. Lycopene β-cyclases are found in a wide range of sources, mainly plants and microorganisms. Lycopene β-cyclases have been extensively studied for their important catalytic activity, including for use in genetic engineering to modify plants and microorganisms, as a blocking target for lycopene industrial production strains, and for their genetic and physiological effects related to microorganic and plant biological traits. This review of lycopene β-cyclases summarizes the major studies on their basic classification, functional activity, metabolic engineering, and plant science.

Converting Escherichia coli MG1655 into a chemical overproducer through inactivating defense system against exogenous DNA

Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production. However, the direct application of this strain suffers from the transformation inefficiency and plasmid instability. Herein, we conducted genetic modifications at a serial of loci of MG1655 genome, generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways. Using JW128 as the host, the titer of isobutanol reached 5.76 g/L in shake-flask fermentation, and the titer of lycopene reached 1.91 g/L in test-tube fermentation, 40-fold and 5-fold higher than that of original MG1655, respectively. These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.

Fast and antibiotic free genome integration into Escherichia\xa0coli chromosome

Genome-based Escherichia coli expression systems are superior to conventional plasmid-based systems as the metabolic load triggered by recombinant compounds is significantly reduced. The efficiency of T7-based transcription compensates for low gene dosage (single copy) and facilitates high product formation rates. While common Gene Bridges’ λ-red mediated recombination technique for site directed integration of genes into the host genome is very efficient, selection for positive clones is based on antibiotic resistance markers and removal thereof is often time consuming. For the generation of industrial production strains, flexibility in terms of integration site is not required, yet time from gene design to a stable clone is a quite relevant parameter. In this study, we developed a fast, efficient and antibiotic-free integration method for E. coli as production strain. We combined the λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. In a first step, λ-red proteins are performing genome integration of a linear, antibiotic marker-free integration cassette. The engineered host strain carries the I-SceI restriction sequence at the attTn7 site, where the integration event happens. After homologous recombination and integration at the target site, site-specific genome cleavage by endonuclease I-SceI is induced, thereby killing all cells still containing an intact I-SceI site. In case of positive recombination events, the genomic I-SceI site is deleted and cleavage is no longer possible. Since plasmids are designed to contain another I-SceI restriction site they are destroyed by self-cleavage, a procedure replacing the time-consuming plasmid curing. The new plasmid-based “All-In-One” genome integration method facilitates significantly accelerated generation of genome-integrated production strains in 4 steps.

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to ~ 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains.

High-Density pH-Auxostat Fed-Batch Culture of Schizochytrium limacinum SR21 with Acetic Acid as a Carbon Source

Schizochytrium limacinum SR21 is an important strain for industrial production of docosahexaenoic acid (DHA), which is an important omega-3 fatty acid used in the nutraceutical and food industry. However, the high cost of carbon sources has limited its further application in the market with much larger volume, such as animal feed for aquaculture, poultry, and livestock. To seek low-cost carbon source, acetic acid is tested in the present study. The effect of different factors, including initial carbon source concentration, pH, aeration rate, and nitrogen sources, on biomass, lipid, and DHA production were tested. With optimized culture conditions, the biomass concentration of 146g/L, total fatty acids (TFAs) of 82.3g/L, and DHA content of 23.0g/L were achieved with a pH-auxostat fed-batch cultivation. These results suggested that acetic acid is a promising feedstock for the low-cost production of DHA. Graphical Abstract.

Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design

The development of Pseudomonas strains for industrial production of fuels and chemicals will require the integration of heterologous genes and pathways into the chromosome. Finding the most appropriate integration site to maximize strain performance is an essential part of the strain design process. We characterized seven chromosomal loci in Pseudomonas putida KT2440 for integration of a fluorescent protein expression construct. Insertion in five of the loci did not affect growth rate, but fluorescence varied by up to 27-fold. Three sites displaying a diversity of phenotypes with the fluorescent reporter were also chosen for the integration of a gene encoding a muconate importer. Depending on the integration locus, expression of the importer varied by approximately 3-fold and produced significant phenotypic differences. This work demonstrates the impact of the integration location on host viability, gene expression, and overall strain performance.

Enhanced production of bacitracin via energy metabolism engineering in Bacillus licheniformis DW2

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.

Development of optimal steam explosion pretreatment and highly effective cell factory for bioconversion of grain vinegar residue to butanol

BackgroundThe industrial vinegar residue (VR) from solid-state fermentation, mainly cereals and their bran, will be a potential feedstock for future biofuels because of their low cost and easy availability. However, utilization of VR for butanol production has not been as much optimized as other sources of lignocellulose, which mainly stem from two key elements: (i) high biomass recalcitrance to enzymatic sugar release; (ii) lacking of suitable industrial biobutanol production strain. Though steam explosion has been proved effective for bio-refinery, few studies report SE for VR pretreatment. Much of the relevant knowledge remains unknown. Meanwhile, recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strain are quite limited. In this study, we assessed the impact of SE pretreatment, enzymatic hydrolysis kinetics, overall sugar recovery and applied atmospheric and room temperature plasma (ARTP) mutant method for the Clostridium strain development to solve the long-standing problem.ResultsSE pretreatment was first performed. At the optimal condition, 29.47% of glucan, 71.62% of xylan and 22.21% of arabinan were depolymerized and obtained in the water extraction. In the sequential enzymatic hydrolysis process, enzymatic hydrolysis rate was increased by 13-fold compared to the VR without pretreatment and 19.60 g glucose, 15.21 g xylose and 5.63 g arabinose can be obtained after the two-step treatment from 100 g VR. Porous properties analysis indicated that steam explosion can effectively generate holes with diameter within 10–20 nm. Statistical analysis proved that enzymatic hydrolysis rate of VR followed the Pseudop-second-order kinetics equation and the relationship between SE severity and enzymatic hydrolysis rate can be well revealed by Boltzmann model. Finally, a superior inhibitor-tolerant strain, Clostridium acetobutylicum Tust-001, was generated with ARTP treatment. The water extraction and enzymolysis liquid gathered were successfully fermented, resulting in butanol titer of 7.98 g/L and 12.59 g/L of ABE.ConclusionsSE proved to be quite effective for VR due to high fermentable sugar recovery and enzymatic hydrolysate fermentability. Inverse strategy employing ARTP and repetitive domestication for strain breeding is quite feasible, providing us with a new tool for solving the problem in the biofuel fields.

Application of the cbh1 promoter in Mortierella alpina and optimization of induction conditions.

Mortierella alpina has gained remarkable interest due to its high capacity for arachidonic acid (AA) production and potential for eicosapentaenoic acid (EPA) production recently. However, the development of genetically modified strains is limited by lacking inducible promoters, which can express genes conditionally. Here the inducible promoter of cellobiohydrolase (Pcbh1) was utilized in M. alpina and the gene oPpFADS17 encoding ω-3 fatty acid desaturase was selected as the reporter gene. Under conditions with inducer, expression of this gene enables M. alpina to produce EPA at room temperature, while no EPA was detected without inducer. We then optimized the induction conditions. The results demonstrated that the optimal induction condition was broth medium with 1% avicel as the inducer and 5% glucose as extra carbon source and the transcription level of the reporter gene was increasing with the extension of induction time. Successful application of Pcbh1 in M. alpina would significantly contribute to the steerable system to construct engineered strains for industrial production of microbial oils. SIGNIFICANCE AND IMPACT OF THE STUDY: Mortierella alpina is a commercial strain for production of polyunsaturated fatty acids. Genetic engineering strategies based on M. alpina require the development of inducible promoters to regulate gene expression conditionally at specific times. However, available inducible promoters for M. alpina were limited. In this study, we explore the feasibility of inducible cbh1 promoter in M. alpina and determined the optimal induction condition, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of the reporter gene under the control of Pcbh1 showed that Pcbh1 is a strong inducible promoter for M. alpina.

Engineering Cell Wall Integrity Enables Enhanced Squalene Production in Yeast.

Microbial production of many lipophilic compounds is often limited by product toxicity to host cells. Engineering cell walls can help mitigate the damage caused by lipophilic compounds by increasing tolerance to those compounds. To determine if the cell wall engineering would be effective in enhancing lipophilic compound production, we used a previously constructed squalene-overproducing yeast strain (SQ) that produces over 600 mg/L of squalene, a model membrane-damaging lipophilic compound. This SQ strain had significantly decreased membrane rigidity, leading to increased cell lysis during fermentation. The SQ strain was engineered to restore membrane rigidity by activating the cell wall integrity (CWI) pathway, thereby further enhancing its squalene production efficiency. Maintenance of CWI was associated with improved squalene production, as shown by cell wall remodeling through regulation of Ecm33, a key regulator of the CWI pathway. Deletion of ECM33 in the SQ strain helped restore membrane rigidity and improve stress tolerance. Moreover, ECM33 deletion suppressed cell lysis and increased squalene production by approximately 12% compared to that by the parent SQ strain. Thus, this study shows that engineering of the yeast cell wall is a promising strategy for enhancing the physiological functions of industrial strains for production of lipophilic compounds.

Overexpression of wysR gene enhances wuyiencin production in ΔwysR3 mutant strain of Streptomyces albulus var. wuyiensis strain CK-15.

The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK-15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin-resistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant. We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development. Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.

Reconstitution of TCA cycle involving l-isoleucine dioxygenase for hydroxylation of l-isoleucine in Escherichia coli using CRISPR-Cas9.

l-isoleucine dioxygenase (IDO) is an Fe (II)/α-ketoglutarate (α-KG)-dependent dioxygenase that specifically converts l-isoleucine (l-Ile) to (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is an important drug for the treatment and prevention of type 1 and type 2 diabetes but the yields using current methods are low. In this study, the CRISPR-Cas9 gene editing system was used to knockout sucAB and aceAK gene in the TCA cycle pathway of Escherichia coli (E. coli). For single-gene knockout, the whole process took approximately 7days. However, the manipulation time was reduced by 2days for each round of gene modification for multigene editing. Using the genome-edited recombinant strain E. coli BL21(DE3) ΔsucABΔaceAK/pET-28a(+)-ido (2Δ-ido), the bioconversion ratio of L-Ile to 4-HIL was enhanced by about 15% compared to E. coli BL21(DE3)/pET-28a(+)-ido [BL21(DE3)-ido] strain. The CRISPR-Cas9 editing strategy has the potential in modifying multiple genes more rapidly and in optimizing strains for industrial production.

Metabolic engineering of an industrial Aspergillus niger strain for itaconic acid production

Itaconic acid is a value-added organic acid that is widely applied in industrial production. It can be converted from citric acid by some microorganisms including Aspergillus terreus and Aspergillus niger. Because of high citric acid production (more than 200g/L), A. niger strains may be developed into powerful itaconic acid-producing microbial cell factories. In this study, industrial citric acid-producing strain A. niger YX-1217, capable of producing 180.0-200.0g/L, was modified to produce itaconic acid by metabolic engineering. A key gene cadA encoding aconitase was expressed in A. niger YX-1217 under the control of three different promoters. Analyses showed that the PglaA promoter resulted in higher levels of gene expression than the PpkiA and PgpdA promoters. Moreover, the synthesis pathway of itaconic acid was extended by introducing the acoA gene, and the cadA gene, encoding aconitate decarboxylase, into A. niger YX-1217 under the function of the two rigid short-peptide linkers L1 or L2. The resulting recombinant strains L-1 and L-2 were induced to produce itaconic acid in fed-batch fermentations under three-stage control of agitation speed. After fermentation for 104h, itaconic acid concentrations in the recombinant strain L-2 culture reached 7.2g/L, which represented a 71.4% increase in itaconic acid concentration compared with strain Z-17 that only expresses cadA. Therefore, co-expression of acoA and cadA resulted in an extension of the citric acid metabolic pathway to the itaconic acid metabolic pathway, thereby increasing the production of itaconic acid by A. niger.