[Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum].

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter PgpmA* from six candidate promoters for expressing ilvBN. PgpmA* showcased the expression strength 23.3-fold that of the native promoter PilvBN. Moreover, three synthetic RBS libraries based on the promoter PgpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that "R(9)N(6)" was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of PilvBN. Finally, the promoter PgpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBNS155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Branched-chain amino acids: physico-chemical properties, industrial synthesis and role in signaling, metabolism and energy production.

Branched-chain amino acids (BCAAs)-leucine (Leu), isoleucine (Ile), and valine (Val)-are essential nutrients with significant roles in protein synthesis, metabolic regulation, and energy production. This review paper offers a detailed examination of the physico-chemical properties of BCAAs, their industrial synthesis, and their critical functions in various biological processes. The unique isomerism of BCAAs is presented, focusing on analytical challenges in their separation and quantification as well as their solubility characteristics, which are crucial for formulation and purification applications. The industrial synthesis of BCAAs, particularly using bacterial strains like Corynebacterium glutamicum, is explored, alongside methods such as genetic engineering aimed at enhancing production, detailing the enzymatic processes and specific precursors. The dietary uptake, distribution, and catabolism of BCAAs are reviewed as fundamental components of their physiological functions. Ultimately, their multifaceted impact on signaling pathways, immune function, and disease progression is discussed, providing insights into their profound influence on muscle protein synthesis and metabolic health. This comprehensive analysis serves as a resource for understanding both the basic and complex roles of BCAAs in biological systems and their industrial application.

The ilv2 gene, encoding acetolactate synthase for branched chain amino acid biosynthesis, is required for plant pathogenicity by Leptosphaeria maculans.

BACKGROUND: Control of blackleg disease of canola caused by the fungus Leptosphaeria maculans relies on strategies such as the inhibition of growth with fungicides. However, other chemicals are used during canola cultivation, including fertilizers and herbicides. There is widespread use of herbicides that target the acetolactate synthase (ALS) enzyme involved in branched chain amino acid synthesis and low levels of these amino acids within leaves of Brassica species. In L. maculans the ilv2 gene encodes ALS and thus ALS-inhibiting herbicides may inadvertently impact the fungus. METHODS AND RESULTS: Here, the impact of a commercial herbicide targeting ALS and mutation of the homologous ilv2 gene in L. maculans was explored. Exposure to herbicide had limited impact on growth in vitro but reduced lesion sizes in plant disease experiments. Furthermore, the mutation of the ilv2 gene via CRISPR-Cas9 gene editing rendered the fungus non-pathogenic. CONCLUSION: Herbicide applications can influence disease outcome, but likely to a minor extent.

Duplication and Functional Divergence of Branched-Chain Amino Acid Biosynthesis Genes in Aspergillus nidulans.

Fungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding ß-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both ß-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)2Cys6 transcription factor LeuB. IMPORTANCE Branched-chain amino acid (BCAA) biosynthesis is important for pathogenic fungi to successfully cause disease in human and plant hosts. The enzymes for their production are absent from humans and, therefore, provide potential antifungal targets. While BCAA biosynthesis is well characterized in yeasts, it is poorly understood in filamentous fungal pathogens. Developing a thorough understanding of both the genes encoding the metabolic enzymes for BCAA biosynthesis and how their expression is regulated will inform target selection for antifungal drug development.

Multiple concurrent and convergent stages of genome reduction in bacterial symbionts across a stink bug family.

Nutritional symbioses between bacteria and insects are prevalent and diverse, allowing insects to expand their feeding strategies and niches. A common consequence of long-term associations is a considerable reduction in symbiont genome size likely influenced by the radical shift in selective pressures as a result of the less variable environment within the host. While several of these cases can be found across distinct insect species, most examples provide a limited view of a single or few stages of the process of genome reduction. Stink bugs (Pentatomidae) contain inherited gamma-proteobacterial symbionts in a modified organ in their midgut and are an example of a long-term nutritional symbiosis, but multiple cases of new symbiont acquisition throughout the history of the family have been described. We sequenced the genomes of 11 symbionts of stink bugs with sizes that ranged from equal to those of their free-living relatives to less than 20%. Comparative genomics of these and previously sequenced symbionts revealed initial stages of genome reduction including an initial pseudogenization before genome reduction, followed by multiple stages of progressive degeneration of existing metabolic pathways likely to impact host interactions such as cell wall component biosynthesis. Amino acid biosynthesis pathways were retained in a similar manner as in other nutritional symbionts. Stink bug symbionts display convergent genome reduction events showing progressive changes from a free-living bacterium to a host-dependent symbiont. This system can therefore be used to study convergent genome evolution of symbiosis at a scale not previously available.

The chemical mechanisms of the enzymes in the branched-chain amino acids biosynthetic pathway and their applications.

l-Valine, l-isoleucine, and l-leucine are three key proteinogenic amino acids, and they are also the essential amino acids required for mammalian growth, possessing important and to some extent, special physiological and biological functions. Because of the branched structures in their carbon chains, they are also named as branched-chain amino acids (BCAAs). This review will highlight the advance in studies of the enzymes involved in the biosynthetic pathway of BCAAs, concentrating on their chemical mechanisms and applications in screening herbicides and antibacterial agents. The uses of some of these enzymes in lab scale organic synthesis are also discussed.

An Autoimmunogenic and Proinflammatory Profile Defined by the Gut Microbiota of Patients With Untreated Systemic Lupus Erythematosus.

OBJECTIVE: Changes in gut microbiota have been linked to systemic lupus erythematosus (SLE), but knowledge is limited. Our study aimed to provide an in-depth understanding of the contribution of gut microbiota to the immunopathogenesis of SLE. METHODS: Fecal metagenomes from 117 patients with untreated SLE and 52 SLE patients posttreatment were aligned with 115 matched healthy controls and analyzed by whole-genome profiling. For comparison, we assessed the fecal metagenome of MRL/lpr mice. The oral microbiota origin of the gut species that existed in SLE patients was documented by single-nucleotide polymorphism-based strain-level analyses. Functional validation assays were performed to demonstrate the molecular mimicry of newly found microbial peptides. RESULTS: Gut microbiota from individuals with SLE displayed significant differences in microbial composition and function compared to healthy controls. Certain species, including the Clostridium species ATCC BAA-442 as well as Atopobium rimae, Shuttleworthia satelles, Actinomyces massiliensis, Bacteroides fragilis, and Clostridium leptum, were enriched in SLE gut microbiota and reduced after treatment. Enhanced lipopolysaccharide biosynthesis aligned with reduced branched chain amino acid biosynthesis was observed in the gut of SLE patients. The findings in mice were consistent with our findings in human subjects. Interestingly, some species with an oral microbiota origin were enriched in the gut of SLE patients. Functional validation assays demonstrated the proinflammatory capacities of some microbial peptides derived from SLE-enriched species. CONCLUSION: This study provides detailed information on the microbiota of untreated patients with SLE, including their functional signatures, similarities with murine counterparts, oral origin, and the definition of autoantigen-mimicking peptides. Our data demonstrate that microbiome-altering approaches may offer valuable adjuvant therapies in SLE.

The anaerobic survival mechanism of Schizophyllum commune 20R-7-F01, isolated from deep sediment 2 km below the seafloor.

Fungi dominated the eukaryotic group in the anaerobic sedimentary environment below the ocean floor where they play an essential ecological role. However, the adaptive mechanism of fungi to these anaerobic environments is still unclear. Here, we reported the anaerobic adaptive mechanism of Schizophyllum commune 20R-7-F01, isolated from deep coal-bearing sediment down to ~2 km below the seafloor, through biochemical, metabolomic and transcriptome analyses. The fungus grows well, but the morphology changes obviously and the fruit body develops incompletely under complete hypoxia. Compared with aerobic conditions, the fungus has enhanced branched-chain amino acid biosynthesis and ethanol fermentation under anaerobic conditions, and genes related to these metabolisms have been significantly up-regulated. Additionally, the fungus shows novel strategies for synthesizing ethanol by utilizing both glycolysis and ethanol fermentation pathways. These findings suggest that the subseafloor fungi may adopt multiple mechanisms to cope with lack of oxygen.

The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii.

Metarhizium spp. are well-known biocontrol agents used worldwide to control different insect pests. Keto-acid reductoisomerase (ILVC) is a key enzyme for branched-chain amino acid (BCAA) biosynthesis, and it regulates many physiological activities. However, its functions in insect-pathogenic fungi are poorly understood. In this work, we identified MrilvC in M. robertsii and dissected its roles in fungal growth, conidiation, germination, destruxin biosynthesis, environmental stress response, and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidial germination of an MrilvC-deficient strain. Further feeding assays with intermediates showed that some conidia of the ΔMrilvC mutant start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in the ΔMrilvC mutant. In conclusion, we found intermediates in BCAA biosynthesis are indispensable for Metarhizium robertsii conidial germination. This study will advance our understanding of the fungal germination mechanism.IMPORTANCE Branched-chain amino acid (BCAA) metabolism plays a significant role in many biological activities beyond protein synthesis. Spore germination initiates the first stage of vegetative growth, which is critical for the virulence of pathogenic fungi. In this study, we demonstrated that the keto-acid reductoisomerase MrILVC, a key enzyme for BCAA biosynthesis, from the insect-pathogenic fungus Metarhizium robertsii is associated with conidial germination and fungal pathogenicity. Surprisingly, the germination of the ΔMrilvC mutant was restored when supplemented with the intermediates of BCAA metabolism rather than three BCAAs. The result was significantly different from that of plant-pathogenic fungi. Therefore, this report highlights that the intermediates in BCAA biosynthesis are indispensable for conidial germination of M. robertsii.

Structures of fungal and plant acetohydroxyacid synthases.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.

Inactivation of the AMPK-GATA3-ECHS1 Pathway Induces Fatty Acid Synthesis That Promotes Clear Cell Renal Cell Carcinoma Growth.

The tumorigenic role and underlying mechanisms of lipid accumulation, commonly observed in many cancers, remain insufficiently understood. In this study, we identified an AMP-activated protein kinase (AMPK)-GATA-binding protein 3 (GATA3)-enoyl-CoA hydratase short-chain 1 (ECHS1) pathway that induces lipid accumulation and promotes cell proliferation in clear cell renal cell carcinoma (ccRCC). Decreased expression of ECHS1, which is responsible for inactivation of fatty acid (FA) oxidation and activation of de novo FA synthesis, positively associated with ccRCC progression and predicted poor patient survival. Mechanistically, ECHS1 downregulation induced FA and branched-chain amino acid (BCAA) accumulation, which inhibited AMPK-promoted expression of GATA3, a transcriptional activator of ECHS1. BCAA accumulation induced activation of mTORC1 and de novo FA synthesis, and promoted cell proliferation. Furthermore, GATA3 expression phenocopied ECHS1 in predicting ccRCC progression and patient survival. The AMPK-GATA3-ECHS1 pathway may offer new therapeutic approaches and prognostic assessment for ccRCC in the clinic. SIGNIFICANCE: These findings uncover molecular mechanisms underlying lipid accumulation in ccRCC, suggesting the AMPK-GATA3-ECHS1 pathway as a potential therapeutic target and prognostic biomarker.

Mechanism and Inhibitor Exploration with Binuclear Mg Ketol-Acid Reductoisomerase: Targeting the Biosynthetic Pathway of Branched-Chain Amino Acids.

Binuclear Mg ketol-acid reductoisomerase (KARI), which converts (S)-2-acetolactate into (R)-2,3-dihydroxyisovalerate, is responsible for the second step of the biosynthesis of branched-chain amino acids in plants and microorganisms and thus serves as a key inhibition target potentially without effects on mammals. Here, through the use of density functional calculations and a chemical model, the KARI-catalyzed reaction has been demonstrated to include the initial deprotonation of the substrate C2 hydroxy group, bridged by the two Mg ions, alkyl migration from the C2-alkoxide carbon atom to the C3-carbonyl carbon atom, and hydride transfer from a nicotinamide adenine dinucleotide phosphate [NAD(P)H] cofactor to C2. A dead-end mechanism with a hydride transferred to the C3 carbonyl group has been ruled out. The nucleophilicity (migratory aptitude) of the migrating carbon atom and the provision of additional negative charge to the di-Mg coordination sphere have significant effects on the steps of alkyl migration and hydride transfer, respectively. Other important mechanistic characteristics are also revealed. Inspired by the mechanism, an inhibitor (2-carboxylate-lactic acid) was designed and predicted by barrier analysis to be effective in inactivating KARI, hence probably enriching the antifungal and antibacterial library. Two types of slow substrate analogues (2-trihalomethyl acetolactic acids and 2-glutaryl lactic acid) were also found.

Derepression of bkd by the FadR loss dictates elevated production of BCFAs and isoleucine starvation.

In many γ-proteobacteria, FadR is recognized as a global transcriptional regulator: in addition to being the most prominent regulator for FA biosynthesis and degradation, the protein also mediates expression of many genes in diverse biological processes. In Shewanella oneidensis, a bacterium renowned for its respiratory versatility, FadR directly controls only a few genes. However, the FadR loss substantially increases BCFA contents and impairs growth. In this study, we showed that FadR is required to activate a number of important FA biosynthesis genes, including fabA, fabB, and fabH1. Although most of these genes are controlled by FadR in a direct manner, they are not critically responsible for the phenotypes resulting from the FadR depletion. Subsequent investigations identified BKD encoded by the bkd operon as the critical factor for enhanced BCFA production. In the absence of FadR, the bkd operon is derepressed, resulting in elevated conversion of 3MOP to 3-methylbutanoyl-CoA, one of the direct substrates for BCFA synthesis. We further showed that the growth defect of the fadR mutant is due to BCAA shortage, a scenario also attributable to excessive BKD: 3MOP, the common substrate for both BCFA and BCAA, is disproportionately used for BCFA synthesis, leading to reduced production of BCAA. Collectively, our findings reveal that the S. oneidensis FadR regulon is surely larger than previously proposed and a new mechanism by which FadR impacts bacterial physiology.

Comparative metabolomic analysis reveals different evolutionary mechanisms for branched-chain amino acids production.

Evolution is a powerful tool for the breeding of microorganisms, while the connection between the changes of intracellular metabolism and different evolution directions is still unclear, which once clarified, will greatly expand the application of evolutionary engineering. We aim to clarify the correlation between metabolism changes and evolution directions in two Corynebacterium glutamicum strains for L-valine and L-leucine overproducing originated from the same parental strain by repeated random mutagenesis and selection. GC-MS metabolomics was performed to identify and quantify intracellular metabolites of the evolved and wild-type C. glutamicum strains. Time-series comparison of the fermentation processes was performed. The metabolism differences of three strains mainly exist in central carbon metabolism and the stress-resisting modes. C. glutamicum XV developed an overall "pyruvate-saving" mode for L-valine synthesis, and adopted a trehalose accumulating strategy to resist environmental stresses. C. glutamicum CP depended on an enhanced "pyruvate-producing" mode, together with certain "pyruvate-saving" strategies, for efficient L-leucine synthesis, and accumulated proline, my-inositol, and inositol as the stress-resisting measure. These elaborate regulation strategies could be used in future metabolic engineering, making evolution more informative and applicable.

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 active site of the Mycobacterium tuberculosis branched-chain amino acid biosynthesis enzyme dihydroxyacid dehydratase contains a 2Fe-2S cluster.

Iron-sulfur clusters are protein cofactors with an ancient evolutionary origin. These clusters are best known for their roles in redox proteins such as ferredoxins, but some iron-sulfur clusters have nonredox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize. Here we report a structural and functional characterization of dihydroxyacid dehydratase (DHAD) from Mycobacterium tuberculosis (Mtb), an essential enzyme in the biosynthesis of branched-chain amino acids. Conducting this analysis under fully anaerobic conditions, we solved the DHAD crystal structure, at 1.88 Å resolution, revealing a 2Fe-2S cluster in which one iron ligand is a potentially exchangeable water molecule or hydroxide. UV and EPR spectroscopy both suggested that the substrate binds directly to the cluster or very close to it. Kinetic analysis implicated two ionizable groups in the catalytic mechanism, which we postulate to be Ser-491 and the iron-bound water/hydroxide. Site-directed mutagenesis showed that Ser-491 is essential for activity, and substrate docking indicated that this residue is perfectly placed for proton abstraction. We found that a bound Mg2+ ion 6.5 Å from the 2Fe-2S cluster plays a key role in substrate binding. We also identified a putative entry channel that enables access to the cluster and show that Mtb-DHAD is inhibited by a recently discovered herbicide, aspterric acid, that, given the essentiality of DHAD for Mtb survival, is a potential lead compound for the design of novel anti-TB drugs.

FgIlv3a is crucial in branched-chain amino acid biosynthesis, vegetative differentiation, and virulence in Fusarium graminearum.

Dihydroxyacid dehydratase (DHAD), encoded by ILV3, catalyses the third step in the biosynthetic pathway of branched-chain amino acids (BCAAs), which include isoleucine (Ile), leucine (Leu), and valine (Val). Enzymes involved in BCAA biosynthesis exist in bacteria, plants, and fungi but not in mammals and are therefore attractive targets for antimicrobial or herbicide development. In this study, three paralogous ILV3 genes (FgILV3A, FgILV3B, and FgILV3C) were identified in the genome of Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Deletion of FgILV3A alone or combined with FgILV3B or FgILV3C indicated an important role for FgILV3A in BCAA biosynthesis. FgILV3A deletion mutants lost the ability to grow on medium lacking amino acids. Exogenous supplementation of 1 mM Ile and Val rescued the auxotrophy of ΔFgIlv3A, though 5 mM was required to recover the growth defects in ΔFgIlv3AB and ΔFgIlv3AC strains, indicating that FgIlv3b and FgIlv3c exhibit redundant but accessory roles with FgIlv3a in BCAA biosynthesis. The auxotrophy of ΔFgIlv3A resulted in pleiotropic defects in aerial hyphal growth, in conidial formation and germination, and in aurofusarin accumulation. In addition, the mutants showed reduced virulence and deoxynivalenol production. Overall, our study demonstrates that FgIlv3a is crucial for BCAA biosynthesis in F. graminearum and a candidate fungicide target for FHB management.

Understanding the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation based on integration of RNA-Seq and metabolite data.

The thermotolerant Kluyveromyces marxianus is a potential candidate for high-temperature ethanol fermentation. Although K. marxianus exhibited high ethanol productivity at 45 °C during the early fermentation stage, we observed a fermentation arrest due to the accumulated inhibitors. The stress responses of K. marxianus during high-temperature fermentation were revealed based on integration of RNA sequencing (RNA-Seq) and metabolite data. High temperature stimulated mitochondrial respiration but repressed the tricarboxylic acid (TCA) cycle, leading to increased generation of reactive oxygen species (ROS) and a lowered ratio of reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+). Glycerol production was enhanced during the early fermentation stage, which might contribute to NADH reoxidation and ROS generation. Excess ROS could be neutralized by reduced nicotinamide adenine dinucleotide phosphate (NADPH) that might be reserved in the following ways: (1) decreased biosynthesis of branched-chain amino acids (BCAAs) reduced NADPH consumption; (2) enhanced acetic acid production increased NADPH regeneration. The degree of fatty acid unsaturation was also reduced to adapt to high temperature. In addition, stress responses were also observed after the fermentation arrest at 45 °C. Genes related to peroxidase activity, iron-sulfur cluster assembly, and flavin mononucleotide (FMN) binding were downregulated, while genes associated with DNA repair and lipid composition of the plasma were upregulated. The yeast also produced more ergosterol to deal with ethanol stress. This study gains comprehensive insights into the K. marxianus transcriptome under various stresses during high-temperature ethanol fermentation, providing rich information for further metabolic engineering towards improved stress tolerance and ethanol production.

Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases.

The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn't, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases.

Synthesis of non-canonical branched-chain amino acids in Escherichia coli and approaches to avoid their incorporation into recombinant proteins.

In E. coli the non-canonical amino acids acids norvaline, norleucine, and ß-methylnorleucine, which derive from an off-pathway of the branched-chain amino acid synthesis route are synthesized and incorporated into cellular and recombinant proteins. The synthesis of these amino acids is supported by a high flux of glucose through the glycolytic pathway in combination with a derepression of the enzymes of the branched chain amino acid pathway, for example, when leucine-rich proteins are produced. Avoiding the synthesis and misincorporation of these amino acids has been challenging, especially in large-scale pharmaceutical processes where the problem is boosted by the typical fed-batch production and the technical limitation of mass transfer in the bioreactors. Despite its industrial importance, so far this issue has not been discussed comprehensively. Therefore this paper reviews, firstly, the specific pathway of the non-canonical branched chain amino acids starting at pyruvate, secondly, the molecular factors for their misincorporation, and thirdly, approaches to avoid this misincoporation. While the synthesis of these amino acids is difficult to prevent due to the broad promiscuity of the connected enzymes, recent studies on the control mechanisms of aminoacyl tRNA synthetases open new opportunities to avoid this misincorporation.