Influence of carbon and nitrogen concentration on itaconic acid production by the smut fungus Ustilago maydis

Itaconic acid is a valuable platform compound for the production of bio‐based polymers, chemicals, and fuels. Ustilago maydis is a promising host for the production of itaconic acid from biomass‐derived substrates due to its unicellular growth pattern and its potential to utilize biomass‐derived sugar monomers and polymers. The potential of U. maydis for industrial itaconate production was assessed in pH‐controlled batch fermentations with varying medium compositions. Using 200 g/L glucose and 75 mM ammonium, 44.5 g/L of itaconate was produced at a maximum rate of 0.74 g L^?1 h^?1. By decreasing the substrate concentrations to 50 g/L glucose and 30 mM ammonium, a yield of 0.34 g/g (47 mol%) could be achieved. Itaconate production from xylose was also feasible. These results indicate that high itaconic acid titers can be achieved with U. maydis. However, further optimization of the biocatalyst itself through metabolic engineering is still needed in order to achieve an economically feasible process, which can be used to advance the development of a bio‐based economy.     

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis’ itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5–fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.     

Feeding experiment using uniformly 13C-labeled α-linolenic acid supports the involvement of the decarboxylation mechanism to produce cis-jasmone in Lasiodiplodia theobromae

ABSTRACT

In our previous report, it was found that Lasiodiplodia theobromae produced cis-jasmone via partially utilizing the biosynthetic pathway of JA. A feeding experiment using uniformly ¹³C-labeled α-linolenic acid, which was added to the culture media of the fungus, strongly supported that the fungus produced CJ via the decarboxylation step of the biosynthetic pathway.     

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.     

An Ustilago maydis chassis for itaconic acid production without by-products

Ustilago maydis is a promising yeast for the production of a range of valuable metabolites, including itaconate, malate, glycolipids and triacylglycerols. However, wild-type strains generally produce a potpourri of all of these metabolites, which hinders efficient production of single target chemicals. In this study, the diverse by-product spectrum of U. maydis was reduced through strain engineering using CRISPR/Cas9 and FLP/FRT, greatly increasing the metabolic flux into the targeted itaconate biosynthesis pathway. With this strategy, a marker-free chassis strain could be engineered, which produces itaconate from glucose with significantly enhanced titre, rate and yield. The lack of by-product formation not only benefited itaconate production, it also increases the efficiency of downstream processing improving cell handling and product purity.     

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O‐glycosidically linked to 3,15,16‐trihydroxypalmitic acid. The sugar moiety is acylated with 2‐hydroxy‐octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl‐transferase and is lacking a gene homologous to the α‐hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl‐transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl‐transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.     

Temperature‐dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli

Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two‐stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr^?1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (p_R) and its expression was controlled by the temperature‐sensitive repressor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity, we performed one‐stage and two‐stage bioreactor cultivations. The two‐stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/hr (+48%) and volumetric productivity of 0.39 g/L/hr (+22%) in comparison to the one‐stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L. The temperature‐dependent activation of gene expression by the Lambda promoters (p_R/p_L) has been frequently used to improve protein or, in a few cases, metabolite production in two‐stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock‐down an essential gene (icd) in E. coli to design a two‐stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the potential to be generally used to improve cell factory performance.

     

Diastereoselective hydroxylation and reduction of derivatised tetrahydrofurans by Beauveria bassiana

The filamentous fungus, Beauveria bassiana ATCC 7159, catalyses the regio- and diastereoselective biohydroxylation of trans-2-methyl-5-benzyloxymethyl-tetrahydrofuran to the cis-3-hydroxy derivative. When incubated with cis-2-methyl-3-keto-5-benzyloxymethyltetrahydrofuran, the same fungus performs a reduction to give the cis- and trans-alcohols in a 4:1 ratio.     

Identification of enriched conjugated linoleic acid isomers in cultures of ruminal microorganisms after dosing with 1-<Superscript>13</Superscript>C-linoleic acid

Most studies of linoleic acid biohydrogenation propose that it converts to stearic acid through the production of cis-9 trans-11 CLA and trans-11 C18:1. However, several other CLA have been identified in ruminai contents, suggesting additional pathways may exist. To explore this possibility, this research investigated the linoleic acid biohydrogenation pathway to identify CLA isomers in cultures of ruminai microorganisms after dosing with a ¹³C stable isotope. The ¹³C enrichment was calculated as [(M+1/M)×100] in labeled minus unlabeled cultures. After 48 h incubation, significant ¹³C enrichment was observed in seven CLA isomers, indicating their formation from linoleic acid. All enriched CLA isomers had double bonds in either the 9,11 or 10,12 position except for trans-9 cis-11 CLA. The cis-9 trans-11 CLA exhibited the highest enrichment (30.65%), followed by enrichments from 21.06 to 23.08% for trans-10 cis-12, cis-10 trans-12, trans-9 trans-11, and trans-10 trans-12 CLA. The remaining two CLA (cis-9 cis-11 and cis-10 cis-12 CLA) exhibited enrichments of 18.38 and 19.29%, respectively. The results of this study verified the formation of cis-9 trans-11 and trans-10 cis-12 CLA isomers from linoleic acid biohydrogenation. An additional five CLA isomers also contained carbons originating from linoleic acid, indicating that pathways of linoleic acid biohydrogenation are more complex than previously described.     

Itaconic acid production in microorganisms

Itaconic acid, 2-methylidenebutanedioic acid, is a precursor of polymers, chemicals, and fuels. Many fungi can synthesize itaconic acid; Aspergillus terreus and Ustilago maydis produce up to 85 and 53 g l^?1, respectively. Other organisms, including Aspergillus niger and yeasts, have been engineered to produce itaconic acid. However, the titer of itaconic acid is low compared with the analogous major fermentation product, citric acid, for which the yield is > 200 g l^?1. Here, we review two types of pathway for itaconic acid biosynthesis as well as recent advances by metabolic engineering strategies and process optimization to enhance itaconic acid productivity in native producers and heterologous hosts. We also propose further improvements to overcome existing problems.     

Sequential Depletion and Acquisition of Proteins during Golgi Stack Disassembly and Reformation

Herein, we report the stepwise transport of multiple plant Golgi membrane markers during disassembly of the Golgi apparatus in tobacco leaf epidermal cells in response to the induced expression of the GTP‐locked Sar1p or Brefeldin A (BFA), and reassembly on BFA washout. The distribution of fluorescent Golgi‐resident N‐glycan processing enzymes and matrix proteins (golgins) with specific cis–trans‐Golgi sub‐locations was followed by confocal microscopy during disassembly and reassembly. The first event during Golgi disassembly was the loss of trans‐Golgi enzymes and golgins from Golgi membranes, followed by a sequential redistribution of medial and cis‐Golgi enzymes into the endoplasmic reticulum (ER), whilst golgins were relocated to the ER or cytoplasm. This event was confirmed by fractionation and immuno‐blotting. The sequential redistribution of Golgi components in a trans–cis sequence may highlight a novel retrograde trafficking pathway between the trans‐Golgi and the ER in plants. Release of Golgi markers from the ER upon BFA washout occurred in the opposite sequence, with cis‐matrix proteins labelling Golgi‐like structures before cis/medial enzymes. Trans‐enzyme location was preceded by trans‐matrix proteins being recruited back to Golgi membranes. Our results show that Golgi disassembly and reassembly occur in a highly ordered fashion in plants.     

Enantiopurity and absolute configuration determination of arene cis‐dihydrodiol metabolites and derivatives using chiral boronic acids

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis‐1,2‐diol metabolites, including boronate formation, using racemic or enantiopure (+) and (?)‐2‐(1‐methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono‐ and poly‐cyclic arene cis‐dihydrodiol, cyclohex‐2‐en‐1‐one cis‐diol, heteroarene cis/trans‐2,3‐diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2‐[1‐methoxy‐2,2‐dimethylpropyl]phenyl boronic acid (MDPBA) and 2‐[1‐methoxy‐1‐phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis‐diol metabolites, are presented.     

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Short communication: Benzene metabolism via the intradiol cleavage in a Rhodococcus sp.

Benzene was metabolized by Rhodococcus sp. 33 through the intradiol cleavage (ortho-) pathway producing cis-benzene glycol, catechol and cis, cis-muconic acid as the intermediates. This is the first elucidation of the pathway by which benzene is degraded by a gram-positive organism. The enzyme assays have also suggested that Rhodococcus 33 does not have a fully functional tricarboxylic acid cycle but may have an operational glyoxylate bypass.     

Biosynthesis of trans-2-hexenal in chloroplasts from Thea sinensis

The biosynthetic pathway of trans-2-hexenal, leaf aldehyde, in isolated chloroplasts of Thea sinensis leaves. was examined using a tracer experiment. A high and specific incorporation of radioactivity into cis-3-hexenal and trans-2-hexenal, was observed when linolenic acid-[U-¹⁴C] was incubated with the isolated chloroplasts. Thus, trans-2-hexenal was biosynthesized via cis-3-hexenal from linolenic acid in the chloroplasts.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.