Discovery, characterization and engineering of ligases for amide synthesis.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

Prolonged Impairment of Short-Chain Fatty Acid and L-Isoleucine Biosynthesis in Gut Microbiome in Patients With COVID-19.

BACKGROUND AND AIMS: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with altered gut microbiota composition. Phylogenetic groups of gut bacteria involved in the metabolism of short chain fatty acids (SCFAs) were depleted in SARS-CoV-2-infected patients. We aimed to characterize a functional profile of the gut microbiome in patients with COVID-19 before and after disease resolution. METHODS: We performed shotgun metagenomic sequencing on fecal samples from 66 antibiotics-naïve patients with COVID-19 and 70 non-COVID-19 controls. Serial fecal samples were collected (at up to 6 times points) during hospitalization and beyond 1 month after discharge. We assessed gut microbial pathways in association with disease severity and blood inflammatory markers. We also determined changes of microbial functions in fecal samples before and after disease resolution and validated these functions using targeted analysis of fecal metabolites. RESULTS: Compared with non-COVID-19 controls, patients with COVID-19 with severe/critical illness showed significant alterations in gut microbiome functionality (P < .001), characterized by impaired capacity of gut microbiome for SCFA and L-isoleucine biosynthesis and enhanced capacity for urea production. Impaired SCFA and L-isoleucine biosynthesis in gut microbiome persisted beyond 30 days after recovery in patients with COVID-19. Targeted analysis of fecal metabolites showed significantly lower fecal concentrations of SCFAs and L-isoleucine in patients with COVID-19 before and after disease resolution. Lack of SCFA and L-isoleucine biosynthesis significantly correlated with disease severity and increased plasma concentrations of CXCL-10, NT- proB-type natriuretic peptide, and C-reactive protein (all P < .05). CONCLUSIONS: Gut microbiome of patients with COVID-19 displayed impaired capacity for SCFA and L-isoleucine biosynthesis that persisted even after disease resolution. These 2 microbial functions correlated with host immune response underscoring the importance of gut microbial functions in SARS-CoV-2 infection pathogenesis and outcome.

Substitution of histone H3 arginine 72 to alanine leads to the deregulation of isoleucine biosynthesis in the budding yeast Saccharomyces cerevisiae.

Histone residues play an essential role in the regulation of various biological processes. In the present study, we utilized the H3/H4 histone mutant library to probe the functional aspects of histone residues in amino acid biosynthesis. We found that the histone residue H3R72 plays a crucial role in the regulation of isoleucine biosynthesis. Substitution of the arginine residue (H3R72) of histone H3 to alanine (H3R72A) renders yeast cells unable to grow in minimal medium. Histone mutant H3R72A requires external supplementation of either isoleucine, serine, or threonine for growth in minimal medium. We also observed that the H3R72 residue and leucine amino acid in synthetic complete medium might play a crucial role in determining the intake of isoleucine and threonine in yeast. Furthermore, gene deletion analysis of ILV1 and CHA1 in the H3R72A mutant confirmed that isoleucine is the sole requirement for growth in minimal medium. Altogether, we have identified that histone H3R72 residue may be crucial for yeast growth in minimal medium by regulating isoleucine biosynthesis through the Ilv1 enzyme in the budding yeast Saccharomyces cerevisiae.

Metabolic engineering of Corynebacterium glutamicum WM001 to improve l-isoleucine production.

In this study, l-isoleucine production in Corynebacterium glutamicum WM001 was improved by deleting three genes in the genome, replacing the native promoter of ilvA in the genome, and overexpression of five genes in an alr-based auxotrophic complementation expression system. The three genes deleted in the genome are alaT, brnQ, and alr. Deletion of alaT improved l-isoleucine production by increasing the supply of pyruvate, whereas deletion of brnQ improved l-isoleucine production by blocking the uptake of extracellular l-isoleucine. Exchange of the native promoter of ilvA with promoter tac or tacM could contribute to l-isoleucine production by increasing 2-ketobutyric acid; tac is better than tacM for improving l-isoleucine yield. Different combinations of the genes ilvBN, ppnK, lrp, and brnFE were overexpressed in an alr-based auxotrophic complementation expression system to further improve l-isoleucine production, and the best yield after 72-H flask fermentation was obtained from the strain WM005/pYCW-1-ilvBN2-ppnK1. Without addition of any antibiotics, WM005/pYCW-1-ilvBN2-ppnK1 could produce 32.1 g/L l-isoleucine after 72-H fed-batch fermentation, which is 34.3% increase compared with the original strain WM001.

Controlled branched-chain amino acids auxotrophy in Listeria monocytogenes allows isoleucine to serve as a host signal and virulence effector.

Listeria monocytogenes (Lm) is a saprophyte and intracellular pathogen. Transition to the pathogenic state relies on sensing of host-derived metabolites, yet it remains unclear how these are recognized and how they mediate virulence gene regulation. We previously found that low availability of isoleucine signals Lm to activate the virulent state. This response is dependent on CodY, a global regulator and isoleucine sensor. Isoleucine-bound CodY represses metabolic pathways including branched-chain amino acids (BCAA) biosynthesis, however under BCAA depletion, as occurs during infection, BCAA biosynthesis is upregulated and isoleucine-unbound CodY activates virulence genes. While isoleucine was revealed as an important input signal, it was not identified how internal levels are controlled during infection. Here we show that Lm regulates BCAA biosynthesis via CodY and via a riboregulator located upstream to the BCAA biosynthesis genes, named Rli60. rli60 is transcribed when BCAA levels drop, forming a ribosome-mediated attenuator that cis-regulates the downstream genes according to BCAA supply. Notably, we found that Rli60 restricts BCAA production, essentially starving Lm, a mechanism that is directly linked to virulence, as it controls the internal isoleucine pool and thereby CodY activity. This controlled BCAA auxotrophy likely evolved to enable isoleucine to serve as a host signal and virulence effector.

Reductive tricarboxylic acid cycle enzymes and reductive amino acid synthesis pathways contribute to electron balance in a Rhodospirillum rubrum Calvin-cycle mutant.

Purple non-sulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Reduced electron carriers not used in biosynthesis must still be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin-cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin-cycle mutant maintains electron balance under such conditions. We detected the reversal of some tricarboxylic acid cycle enzymes, carrying reductive flux from malate or fumarate to αKG. This pathway and the reductive synthesis of αKG-derived amino acids are likely important for electron balance, as supplementing the growth medium with αKG-derived amino acids prevented Rs. rubrum Calvin-cycle-mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin-cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.

The ilvGMEDA Operon Is Regulated by Transcription Attenuation in Vibrio alginolyticus ZJ-T.

Bacteria synthesize amino acids according to their availability in the environment or, in the case of pathogens, within the host. We explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs) (l-leucine, l-valine, and l-isoleucine) in Vibrio alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. In this species, the ilvGMEDA operon encodes the main pathway for biosynthesis of BCAAs. Its upstream regulatory region shows no sequence similarity to the corresponding region in Escherichia coli or other Enterobacteriaceae, and yet we show that this operon is regulated by transcription attenuation. The translation of a BCAA-rich peptide encoded upstream of the structural genes provides an adaptive response similar to the E. coli canonical model. This study of a nonmodel Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.IMPORTANCE This study analyzes the regulation of the biosynthesis of branched-chain amino acids (leucine, valine, and isoleucine) in Vibrio alginolyticus, a marine bacterium that is pathogenic to fish and humans. The results highlight the conservation of the main regulatory mechanism with that of the enterobacterium Escherichia coli, suggesting that such a mechanism appeared early during the evolution of Gram-negative bacteria, allowing adaptation to a wide range of environments.

The σB-dependent regulatory sRNA Rli47 represses isoleucine biosynthesis in Listeria monocytogenes through a direct interaction with the ilvA transcript.

The facultative intracellular pathogen Listeria monocytogenes can persist and grow in a diverse range of environmental conditions, both outside and within its mammalian host. The alternative sigma factor Sigma B (σB) plays an important role in this adaptability and is critical for the transition into the host. While some of the functions of the σB regulon in facilitating this transition are understood the role of σB-dependent small regulatory RNAs (sRNAs) remain poorly characterized. In this study, we focused on elucidating the function of Rli47, a σB-dependent sRNA that is highly induced in the intestine and in macrophages. Using a combination of in silico and in vivo approaches, a binding interaction was predicted with the Shine-Dalgarno region of the ilvA mRNA, which encodes threonine deaminase, an enzyme required for branched-chain amino acid biosynthesis. Both ilvA transcript levels and threonine deaminase activity were increased in a deletion mutant lacking the rli47 gene. The Δrli47 mutant displayed a shorter growth lag in isoleucine-depleted growth media relative to the wild-type, and a similar phenotype was also observed in a mutant lacking σB. The impact of the Δrli47 on the global transcription profile of the cell was investigated using RNA-seq, and a significant role for Rli47 in modulating amino acid metabolism was uncovered. Taken together, the data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis as a way to restrict growth under harsh conditions, potentially contributing to the survival of L. monocytogenes in niches both outside and within the mammalian host.

Strategy for improving L-isoleucine production efficiency in Corynebacterium glutamicum.

As one of the three branched-chain amino acids essential for human body, L-isoleucine is widely used in food, medicine, and feed industries. At present, L-isoleucine is mainly produced by microbial fermentation, and the main production strain is Corynebacterium glutamicum. The biosynthetic pathway of L-isoleucine in C. glutamicum is complex, and the activity of key enzymes and the transcription of key genes in the pathway are strictly regulated. The intracellularly synthesized L-isoleucine is secreted by transporters, and the activity of the transporters is also regulated. These intricate regulatory mechanisms increase the difficulty to engineer the L-isoleucine-producing C. glutamicum. This article focuses on the mechanism of L-isoleucine biosynthesis, secretion, and regulation in C. glutamicum and reviews the various metabolic engineering strategies for improving L-isoleucine production efficiency in C. glutamicum.

Enhancement of substrate supply and ido expression to improve 4-hydroxyisoleucine production in recombinant Corynebacterium glutamicum ssp. lactofermentum.

4-Hydroxyisoleucine (4-HIL) has potential value in treating diabetes. L-isoleucine dioxygenase (IDO) catalyzes the hydroxylation of L-isoleucine (Ile) to form 4-HIL with the concomitant oxidation of α-ketoglutarate (α-KG) and oxygen consumption. In our previous study, by expressing the ido gene in the Ile producer Corynebacterium glutamicum ssp. lactofermentum SN01, 4-HIL was de novo-synthesized from glucose without adding either Ile or α-KG. In this study, synergistically improving the substrates supply and IDO activity was applied to enhance the de novo biosynthesis of 4-HIL. Deletion of aceA and blocking of the glyoxylate pathway effectively enhanced α-KG supply and Ile synthesis and thus improved 4-HIL production to 69.47 ± 2.18 mM, 18.9% higher than in the original strain. Coexpression of mqo with ido further improved Ile synthesis but decreased 4-HIL production, partially due to the inadequate activity of IDO. Coexpression of another gene, ido3, with mqo and ido efficiently promoted IDO activity, thus improving 4-HIL production to 91.54 ± 8.29 mM. Further expression of vgb and promotion of the oxygen uptake rate did not change the 4-HIL titer significantly but increased the 4-HIL production rate in the first 72 h of fermentation. After fermentation in the optimized medium, 4-HIL production by the final strains increased to 112-117 mM, indicating these strains are promising candidates for producing 4-HIL. These results demonstrate that synergistically promoting substrate supply and improving IDO activity are efficient approaches to enhance 4-HIL production in C. glutamicum.

Cysteine synthase A overexpression in Corynebacterium glutamicum enhances l-isoleucine production.

Cysteine synthase A (CysK) catalyzes the last reaction of l-cysteine synthesis in bacteria, but its moonlighting functions have been revealed recently. In this study, CysK was overexpressed in Corynebacterium glutamicum IWJ001, an l-isoleucine producer. Compared with the control IWJ001/pDXW-8, IWJ001/pDXW-8-cysK cells grew fast during log phase, and produced 26.5% more l-isoleucine in flask fermentation and 23.5% more l-isoleucine in fed-batch fermentation. The key genes aspC, lysC, hom, thrB, ilvA, and ilvBN involved in l-isoleucine biosynthesis were all upregulated in IWJ001/pDXW-8-cysK, compared with IWJ001/pDXW-8. In addition, IWJ001/pDXW-8-cysK cells were longer and thicker than IWJ001/pDXW-8 cells. Compared with IWJ001/pDXW-8, the membrane permeability increased 15.8% and biofilm formation ability decreased 71.3% for IWJ001/pDXW-8-cysK cells. The results demonstrate that CysK overexpression in C. glutamicum is a good approach to enhance l-isoleucine production.

High production of 4-hydroxyisoleucine in Corynebacterium glutamicum by multistep metabolic engineering.

4-Hydroxyisoleucine (4-HIL) exhibits a unique glucose-dependent insulinotropic activity and is a promising candidate for the treatment of diabetes. Direct fermentation of 4-HIL has been recently studied; however, the expected titre and yield were not achieved. In this study, we initially developed a pathway for the synthesis of 4-HIL in an L-isoleucine producer, C. glutamicum YI, but insufficient supply of α-ketoglutarate was a bottleneck for a strong production. Six genes involved in oxaloacetate and α-ketoglutarate branches were overexpressed or deleted, which increased the production of 4-HIL to 5.12 g/L but a considerable amount of L-isoleucine still accumulated in the culture. We then dynamically modulated the activity of the α-ketoglutarate dehydrogenase complex (ODHC) by employing L-isoleucine-responsive transcription or attenuation strategies. The best-engineered strain, HIL18, produced 34.21 g/L 4-HIL with a negligible accumulation of byproducts, including approximately 0.6 g/L L-isoleucine. This study achieved the highest production and yield of 4-HIL, and optimizing the TCA cycle by dynamically modulating the activity of ODHA can be a powerful strategy to balance the carbon flux and achieve efficient production of α-ketoglutarate and derivatives.

Biosynthesis and in vitro enzymatic synthesis of the isoleucine conjugate of 12-oxo-phytodienoic acid from the isoleucine conjugate of α-linolenic acid.

The isoleucine conjugate of 12-oxo-phytodienoic acid (OPDA-Ile), a new member of the jasmonate family, was recently identified in Arabidopsis thaliana and might be a signaling molecule in plants. However, the biosynthesis and function of OPDA-Ile remains elusive. This study reports an in vitro enzymatic method for synthesizing OPDA-Ile, which is catalyzed by reactions of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) using isoleucine conjugates of α -linolenic acid (LA-Ile) as the substrate. A. thaliana fed LA-Ile exhibited a marked increase in the OPDA-Ile concentration. LA-Ile was also detected in A. thaliana. Furthermore, stable isotope labelled LA-Ile was incorporated into OPDA-Ile. Thus, OPDA-Ile is biosynthesized via the cyclization of LA-Ile in A. thaliana.

Attempt to simultaneously generate three chiral centers in 4-hydroxyisoleucine with microbial carbonyl reductases.

A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.

Application of a JA-Ile Biosynthesis Inhibitor to Methyl Jasmonate-Treated Strawberry Fruit Induces Upregulation of Specific MBW Complex-Related Genes and Accumulation of Proanthocyanidins.

Fleshy fruits are an important source of anthocyanins and proanthocyanidins (PAs), which protect plants against stress, and their consumption provides beneficial effects for human health. In strawberry fruit, the application of exogenous methyl jasmonate (MeJA) upregulates anthocyanin accumulation, although the relationship between the jasmonate pathway and anthocyanin and PA biosynthesis in fruits remains to be understood. Anthocyanin and PA accumulation is mainly regulated at the transcriptional level through R2R3-MYB and bHLH transcription factors in different plant species and organs. Here, the effect of jarin-1, a specific inhibitor of bioactive JA (jasmonoyl-isoleucine, JA-Ile) biosynthesis, on anthocyanin and PA accumulation was evaluated during strawberry (Fragaria × ananassa) fruit development using an in vitro ripening system for 48 h. Also, we observed the effects of MeJA and the application of jarin-1 to MeJA-treated fruits (MeJA + jarin-1 treatment). We assessed changes of expression levels for the JA-Ile and MeJA biosynthetic (FaJAR1.2 and FaJMT), JA signaling-related (FaMYC2 and FaJAZ1), MYB-bHLH-WD40 (MBW) complex-related (FabHLH3/33, FaMYB9/10/11, and repressor FaMYB1), and anthocyanin and PA biosynthetic (FaANS, FaUFGT, FaANR, and FaLAR) genes. In addition, the promoter region of MBW complex-related MYB genes was isolated and sequenced. We found a higher redness of strawberry fruit skin and anthocyanin content in MeJA-treated fruits with respect to jarin-1-treated ones concomitant with an upregulation of FaANS and FaUFGT genes. Inversely, the PA content was higher in jarin-1- and MeJA + jarin-1-treated than in MeJA-treated fruits. MeJA + jarin-1 treatment resulted in an upregulation of FaANR and associated transcription factors such as FabHLH33 and FaMYB9/11 along with FaJMT and FaJAR1.2. Finally, we found JA-responsive elements in the promoter regions of FaMYB1/9/10/11 genes. It is proposed that PA biosynthesis-related genes can be upregulated by the application of jarin-1 to MeJA-treated fruit, thus increasing PA accumulation in strawberry.

Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.

BACKGROUND: Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking. RESULTS: To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised. CONCLUSIONS: Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Deciphering the Biosynthetic Origin of L-allo-Isoleucine.

The nonproteinogenic amino acid L-allo-isoleucine (L-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and mammalian systems including Homo sapiens. Despite its ubiquity and functional importance, the specific origins of this unique amino acid have eluded characterization. In this study, we describe the discovery and characterization of two enzyme pairs consisting of a pyridoxal 5'-phosphate (PLP)-linked aminotransferase and an unprecedented isomerase synergistically responsible for the biosynthesis of L-allo-Ile from L-isoleucine (L-Ile) in natural products. DsaD/DsaE from the desotamide biosynthetic pathway in Streptomyces scopuliridis SCSIO ZJ46, and MfnO/MfnH from the marformycin biosynthetic pathway in Streptomyces drozdowiczii SCSIO 10141 drive L-allo-Ile generation in each respective system. In vivo gene inactivations validated the importance of the DsaD/DsaE pair and MfnO/MfnH pair in L-allo-Ile unit biosynthesis. Inactivation of PLP-linked aminotransferases DsaD and MfnO led to significantly diminished desotamide and marformycin titers, respectively. Additionally, inactivation of the isomerase genes dsaE and mfnH completely abolished production of all L-allo-Ile-containing metabolites in both biosynthetic pathways. Notably, in vitro biochemical assays revealed that DsaD/DsaE and MfnO/MfnH each catalyze a bidirectional reaction between L-allo-Ile and L-Ile. Site-directed mutagenesis experiments revealed that the enzymatic reaction involves a PLP-linked ketimine intermediate and uses an arginine residue from the C-terminus of each isomerase to epimerize the amino acid ß-position. Consequently, these data provide important new insight into the origins of L-allo-Ile in natural products with medicinal potential and illuminate new possibilities for biotool development.

Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l-isoleucine production.

Three genes, gnd, pgl, and fbp, relevant to the pentose phosphate pathway (PPP) were overexpressed in Corynebacterium glutamicum IWJ001, leading to increase of l-isoleucine production. The transcriptional levels of gnd, pgl, and fbp significantly increased in IWJ001/pDXW-8-gnd-fbp-pgl. Compared with the control strain IWJ001/pDXW-8, intracellular NADPH/NADP+ ratios in IWJ001/pDXW-8-gnd and IWJ001/pDXW-8-gnd-fbp cells grown for 36 H increased threefold and fourfold, respectively, indicating that overexpression of gnd and fbp redirected the carbon flux to PPP. Intracellular NADPH/NADP+ ratio in IWJ001/pDXW-8-gnd-fbp-pgl grown for 36 H was similar to IWJ001/pDXW-8, suggesting that the NADPH produced by PPP could be quickly consumed for l-isoleucine production. 10.9 and 28.96 g/L of l-isoleucine was produced in IWJ001/pDXW-8-gnd-fbp-pgl in shake flask cultivation and fed-batch fermentation, respectively. In addition, IWJ001/pDXW-8-gnd-fbp-pgl grew fast, its dry cell weight reached 49 g/L after 48 H, whereas the start strain IWJ001/pDXW-8 reached only 40 g/L. After 96 H fermentation, l-isoleucine yield on glucose in IWJ001/pDXW-8-gnd-fbp-pgl reached 0.138 g/g. The results demonstrate that carbon flux redirection to PPP is an efficient approach to enhance l-isoleucine production in C. glutamicum.

Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of L-isoleucine biosynthesis.

Previously we have characterized a threonine dehydratase mutant TD(F383V) (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS(P176S, D426E, L575W) (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best L-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK(A279T) (encoded by lysC1) and a homoserine dehydrogenase mutant HD(G378S) (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK(A279T) is completely resistant to feed-back inhibition by L-threonine and L-lysine, and that HD(G378S) is partially resistant to L-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive L-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from L-lysine (decreased by 50.1 %) to L-threonine (4.85 g/L) with minor L-isoleucine and no L-homoserine accumulation, further co-expressing ilvA1 completely depleted L-threonine and strongly shifted carbon flux from L-lysine (decreased by 83.0 %) to L-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD(F383V) might be the main driving force for L-isoleucine over-synthesis in this case, and the partially feed-back resistant HD(G378S) might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.

Involvement of threonine deaminase FgIlv1 in isoleucine biosynthesis and full virulence in Fusarium graminearum.

In this study we characterized FgIlv1, a homologue of the Saccharomyces cerevisiae threonine dehydratase (TD) from the important Fusarium head blight fungus Fusarium graminearum. TD catalyzes the first step in the biosynthesis pathway of isoleucine (Ile) for conversion of threonine (Thr) to 2-ketobutyrate (2-KB). The FgILV1 deletion mutant ΔFgIlv1-3 was unable to grow on minimal medium or fructose gelatin agar which lacked Ile. Exogenous supplementation of Ile or 2-KB but not Thr rescued the mycelial growth defect of ΔFgIlv1-3, indicating the involvement of FgIlv1 in the conversion of Thr to 2-KB in Ile biosynthesis. Additionally, exogenous supplementation of Methionine (Met) could also rescue the mycelial growth defect of ΔFgIlv1-3, indicating a crosstalk between Ile biosynthesis and Met catabolism in F. graminearum. Deletion of FgILV1 also caused defects in conidial formation and germination. In addition, ΔFgIlv1-3 displayed decreased virulence on wheat heads and a low level of deoxynivalenol (DON) production in wheat kernels. Taken together, results of this study indicate that FgIlv1 is an essential component in Ile biosynthesis and is required for various cellular processes including mycelial and conidial morphogenesis, DON biosynthesis, and full virulence in F. graminearum. Our data indicate the potential of targeting Ile biosynthesis for anti-FHB management.