Targets For Metabolic Engineering Research Articles

Evolutionary trajectory of transcription factors and selection of targets for metabolic engineering.

Transcription factors (TFs) provide potentially powerful tools for plant metabolic engineering as they often control multiple genes in a metabolic pathway. However, selecting the best TF for a particular pathway has been challenging, and the selection often relies significantly on phylogenetic relationships. Here, we offer examples where evolutionary relationships have facilitated the selection of the suitable TFs, alongside situations where such relationships are misleading from the perspective of metabolic engineering. We argue that the evolutionary trajectory of a particular TF might be a better indicator than protein sequence homology alone in helping decide the best targets for plant metabolic engineering efforts. This article is part of the theme issue 'The evolution of plant metabolism'.

Regulation of AreA on lipid biosynthesis under different nitrogen sources and C/N ratios in the model oleaginous fungus Mucor circinelloides

Mucor circinelloides has been exploited as model filamentous fungi for studies of genetic manipulation of lipogenesis. It is widely recognized that lipid accumulation is increased when there is a lack of nitrogen source in oleaginous microorganism. Nitrogen metabolism in filamentous fungi is a complex process that can be regulated by the global nitrogen regulator AreA. In this study, we cultivated the areA-knockout and -overexpression strains obtained in our previous study, using 20 different nitrogen sources. It emerged that the disruption of AreA in M. circinelloides reduced its sensitivity to nitrogen availability, resulting in increased lipid synthesis. Specially, the areA-knockout strain was unable to fully utilize many nitrogen sources but the ammonium and glutamate. We continued to investigate lipid production at different molar C/N ratios using glucose as sole carbon source and ammonium sulfate as sole nitrogen source, of which the high C/N ratios activate high lipid accumulation. By comparing the experimental results with transcriptional analysis, we were able to identify the optimal process conditions suitable for lipid accumulation and potential targets for future metabolic engineering.

Biosynthesis and metabolic engineering of isoflavonoids in model plants and crops: a review.

Isoflavonoids, the major secondary metabolites within the flavonoid biosynthetic pathway, play important roles in plant defense and exhibit free radical scavenging properties in mammals. Recent advancements in understanding the synthesis, transport, and regulation of isoflavonoids have identified their biosynthetic pathways as promising targets for metabolic engineering, offering potential benefits such as enhanced plant resistance, improved biomass, and restoration of soil fertility. This review provides an overview of recent breakthroughs in isoflavonoid biosynthesis, encompassing key enzymes in the biosynthetic pathway, transporters influencing their subcellular localization, molecular mechanisms regulating the metabolic pathway (including transcriptional and post-transcriptional regulation, as well as epigenetic modifications). Metabolic engineering strategies aimed at boosting isoflavonoid content in both leguminous and non-leguminous plants. Additionally, we discuss emerging technologies and resources for precise isoflavonoid regulation. This comprehensive review primarily focuses on model plants and crops, offering insights for more effective and sustainable metabolic engineering approaches to enhance nutritional quality and stress tolerance.

CFSA: Comparative flux sampling analysis as a guide for strain design

Genome-scale metabolic models of microbial metabolism have extensively been used to guide the design of microbial cell factories, still, many of the available strain design algorithms often fail to produce a reduced list of targets for improved performance that can be implemented and validated in a step-wise manner. We present Comparative Flux Sampling Analysis (CFSA), a strain design method based on the extensive comparison of complete metabolic spaces corresponding to maximal or near-maximal growth and production phenotypes. The comparison is complemented by statistical analysis to identify reactions with altered flux that are suggested as targets for genetic interventions including up-regulations, down-regulations and gene deletions. We applied CFSA to the production of lipids by Cutaneotrichosporon oleaginosus and naringenin by Saccharomyces cerevisiae identifying engineering targets in agreement with previous studies as well as new interventions. CFSA is an easy-to-use, robust method that suggests potential metabolic engineering targets for growth-uncoupled production that can be applied to the design of microbial cell factories.

Comparative metabolite profiling of salt sensitive Oryza sativa and the halophytic wild rice Oryza coarctata under salt stress.

To better understand the salt tolerance of the wild rice, Oryza coarctata, root tissue-specific untargeted comparative metabolomic profiling was performed against the salt-sensitive Oryza sativa. Under control, O. coarctata exhibited abundant levels of most metabolites, while salt caused their downregulation in contrast to metabolites in O. sativa. Under control conditions, itaconate, vanillic acid, threonic acid, eicosanoids, and a group of xanthin compounds were comparatively abundant in O. coarctata. Similarly, eight amino acids showed constitutive abundance in O. coarctata. In contrast, under control, glycerolipid abundances were lower in O. coarctata and salt stress further reduced their abundance. Most phospholipids also showed a distribution similar to the glycerolipids. Fatty acyls were however significantly induced in O. coarctata but organic acids were prominently induced in O. sativa. Changes in metabolite levels suggest that there was upregulation of the arachidonic acid metabolism in O. coarctata. In addition, the phenylpropanoid biosynthesis as well as cutin, suberin, and wax biosynthesis were also more enriched in O. coarctata, likely contributing to its anatomical traits responsible for salt tolerance. The comparative variation in the number of metabolites like gelsemine, allantoin, benzyl alcohol, specific phospholipids, and glycerolipids may play a role in maintaining the superior growth of O. coarctata in salt. Collectively, our results offer a comprehensive analysis of the metabolite profile in the roots of salt-tolerant O. coarctata and salt-sensitive O. sativa, which confirm potential targets for metabolic engineering to improve salt tolerance and resilience in commercial rice genotypes.

ECMpy 2.0: A Python package for automated construction and analysis of enzyme-constrained models

Genome-scale metabolic models (GEMs) have been widely employed to predict microorganism behaviors. However, GEMs only consider stoichiometric constraints, leading to a linear increase in simulated growth and product yields as substrate uptake rates rise. This divergence from experimental measurements prompted the creation of enzyme-constrained models (ecModels) for various species, successfully enhancing chemical production. Building upon studies that allocate macromolecule resources, we developed a Python-based workflow (ECMpy) that constructs an enzyme-constrained model. This involves directly imposing an enzyme amount constraint in GEM and accounting for protein subunit composition in reactions. However, this procedure demands manual collection of enzyme kinetic parameter information and subunit composition details, making it rather user-unfriendly. In this work, we've enhanced the ECMpy toolbox to version 2.0, broadening its scope to automatically generate ecGEMs for a wider array of organisms. ECMpy 2.0 automates the retrieval of enzyme kinetic parameters and employs machine learning for predicting these parameters, which significantly enhances parameter coverage. Additionally, ECMpy 2.0 introduces common analytical and visualization features for ecModels, rendering computational results more user accessible. Furthermore, ECMpy 2.0 seamlessly integrates three published algorithms that exploit ecModels to uncover potential targets for metabolic engineering. ECMpy 2.0 is available at https://github.com/tibbdc/ECMpy or as a pip package (https://pypi.org/project/ECMpy/).

Saccharomyces cerevisiae as a Host for Chondroitin Production

Chondroitin is a glycosaminoglycan that has gained widespread use in nutraceuticals and pharmaceuticals, mainly for treating osteoarthritis. Traditionally, it has been extracted from animal cartilage but recently, biotechnological processes have emerged as a commercial alternative to avoid the risk of viral or prion contamination and offer a vegan-friendly source. Typically, these methods involve producing the chondroitin backbone using pathogenic bacteria and then modifying it enzymatically through the action of sulfotransferases. Despite the challenges of expressing active sulfotransferases in bacteria, the use of eukaryotic microorganisms is still limited to a few works using Pichia pastoris. To create a safer and efficient biotechnological platform, we constructed a biosynthetic pathway for chondroitin production in S. cerevisiae as a proof-of-concept. Up to 125 mg/L and 200 mg/L of intracellular and extracellular chondroitin were produced, respectively. Furthermore, as genome-scale models are valuable tools for identifying novel targets for metabolic engineering, a stoichiometric model of chondroitin-producing S. cerevisiae was developed and used in optimization algorithms. Our research yielded several novel targets, such as uridine diphosphate (UDP)-N-acetylglucosamine pyrophosphorylase (QRI1), glucosamine-6-phosphate acetyltransferase (GNA1), or N-acetylglucosamine-phosphate mutase (PCM1) overexpression, that might enhance chondroitin production and guide future experimental research to develop more efficient host organisms for the biotechnological production process.

Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.

Enhanced starch accumulation in Chlorella sorokiniana as sugar platform and the expression profiling of key regulatory proteins

Microalgae can accumulate considerable starch as a renewable sugar platform for biofuel production. In this study, Chlorella sorokiniana exhibited a significant increase in starch content from 3.0% to 49.5% DM under sulfur limitation (SL) conditions, and the lower the concentration of sulfur was, the more effective it was. Direct enzymatic hydrolysis of wet biomass resulted in a glucose yield of 97.1%, indicating its potential for fermentable sugar production. However, C. sorokiniana grew slowly, and cell division and biomass accumulation decreased under SL conditions, which could be explained by the decreased photosynthetic efficiency and protein content. The sulfur replenishment (SR) confirmed the rapid response of C. sorokiniana in starch accumulation to sulfur availability. Thus, we further investigated the expression profiling of key regulatory proteins under Control-SL-SR stages. The expression of AGPase A was strongly induced in the SL stage and subsequently downregulated during SR stage, suggesting its role as a key regulatory protein for enhanced starch accumulation. Additionally, downregulation of SBE and GBE expression likely contributed to structural modifications of the starch molecules. These proteins could serve as potential targets for metabolic engineering in further improving the starch production capacity in C. sorokiniana.

FastKnock: an efficient next-generation approach to identify all knockout strategies for strain optimization

Overproduction of desired native or nonnative biochemical(s) in (micro)organisms can be achieved through metabolic engineering. Appropriate rewiring of cell metabolism is performed by making rational changes such as insertion, up-/down-regulation and knockout of genes and consequently metabolic reactions. Finding appropriate targets (including proper sets of reactions to be knocked out) for metabolic engineering to design optimal production strains has been the goal of a number of computational algorithms. We developed FastKnock, an efficient next-generation algorithm for identifying all possible knockout strategies (with a predefined maximum number of reaction deletions) for the growth-coupled overproduction of biochemical(s) of interest. We achieve this by developing a special depth-first traversal algorithm that allows us to prune the search space significantly. This leads to a drastic reduction in execution time. We evaluate the performance of the FastKnock algorithm using various Escherichia coli genome-scale metabolic models in different conditions (minimal and rich mediums) for the overproduction of a number of desired metabolites. FastKnock efficiently prunes the search space to less than 0.2% for quadruple- and 0.02% for quintuple-reaction knockouts. Compared to the classic approaches such as OptKnock and the state-of-the-art techniques such as MCSEnumerator methods, FastKnock found many more beneficial and important practical solutions. The availability of all the solutions provides the opportunity to further characterize, rank and select the most appropriate intervention strategy based on any desired evaluation index. Our implementation of the FastKnock method in Python is publicly available at https://github.com/leilahsn/FastKnock.

Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis.

Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester-forming products all were determined in SA-rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C-3' hydroxylation, and SmCYP98A14 preferentially catalyses C-3 hydroxylation in RA generation. In addition, relative to C-3' hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA-abundant species and versatility of CYP98A enzymes catalytic preference in meta-hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.

Metabolic control analysis enabled the improvement of the L-cysteine production process with Escherichia coli

L-cysteine is an amino acid with relevance to the pharmaceutical, food, feed, and cosmetic industry. The environmental and societal impact of its chemical production has led to the development of more sustainable fermentative L-cysteine production processes with engineered E. coli based on glucose and thiosulfate as sulphur source. Still, most of the published processes show low yields. For the identification of further metabolic engineering targets, engineered E. coli cells were withdrawn from a fed-batch production process, followed by in vivo metabolic control analysis (MCA) based on the data of short-term perturbation experiments, metabolomics (LC–MS), and thermodynamic flux analysis (TFA). In vivo MCA indicated that the activities of the L-cysteine synthases of the cells withdrawn from the production process might be limiting, and we hypothesised that the L-cysteine precursor O-acetylserine (OAS) might be exported from the cells faster than it took to transform OAS into L-cysteine. By increasing the expression of the L-cysteine synthases, either sulfocysteine synthase or L-cysteine synthase, which transform OAS into L-cysteine, an improvement of up to 70% in specific L-cysteine productivity and up to 47% in the final L-cysteine concentration was achieved in standardised fed-batch processes thereby increasing the yield on glucose by more than 85 to 9.2% (w/w).Key points• Metabolic control analysis was applied to analyse L-cysteine production with E. coli• OAS export was faster than its transformation to L-cysteine• Overexpression of L-cysteine synthases improved L-cysteine productivity and yield

Biosynthetic mechanism of the yellow pigments in the marine bacterium Pseudoalteromonas sp. strain T1lg65.

The Pseudoalteromonas genus marine bacteria have attracted increasing interest because of their abilities to produce bioactive metabolites. The pigmented Pseudoalteromonas group encodes more secondary metabolite biosynthetic gene clusters (BGCs) than the non-pigmented group. Here, we report a yellow pigmented bacterium Pseudoalteromonas sp. strain T1lg65, which was isolated from a mangrove forest sediment. We showed that the yellow pigments of T1lg65 belong to the group of lipopeptide alterochromides. Further genetic analyses of the alterochromide BGC revealed that the yellow pigments are biosynthesized by aryl-polyene synthases and nonribosomal peptide synthases. Within the gene cluster, altA encodes a tyrosine ammonia acid lyase, which catalyzes synthesis of the precursor 4-hydroxycinnamic acid (4-HCA) from tyrosine in the alterochromide biosynthetic pathway. In addition, altN, encoding a putative flavin-dependent halogenase, was proven to be responsible for the bromination of alterochromides based on gene deletion, molecular docking, and site mutagenesis analyses. In summary, the biosynthetic pathway, precursor synthesis, and bromination mechanism of the lipopeptide alterochromides were studied in-depth. Our results expand the knowledge on biosynthesis of Pseudoalteromonas pigments and could promote the development of active pigments in the future.IMPORTANCEThe marine bacteria Pseudoalteromonas spp. are important biological resources because they are producers of bioactive natural products, including antibiotics, pigments, enzymes, and antimicrobial peptides. One group of the microbial pigments, alterochromides, holds a great value for their novel lipopeptide structures and antimicrobial activities. Previous studies were limited to the structural characterization of alterochromides and genome mining for the alterochromide biosynthesis. This work focused on the biosynthetic mechanism for alterochromide production, especially revealing functions of two key genes within the gene cluster for the alterochromide biosynthesis. On the one hand, our study provides a target for metabolic engineering of the alterochromide biosynthesis; on the other hand, the 4-HCA synthase AltA and brominase AltN show potential in the biocatalyst industry.

Metabolic Usage and Glycan Destinations of GlcNAz in E. coli.

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars, such as N-acetylglucosamine (GlcNAc), being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N-azidoacetylglucosamine (GlcNAz), for metabolic labeling in Escherichia coli. The cytosolic production of UDP-GlcNAz was confirmed by using fluorescence-assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated was interrogated by analyzing potential sites including peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-β-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS), and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) toward UDP-GlcNAz in vitro and the lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.

Coupling High-Throughput and Targeted Screening for Identification of Nonobvious Metabolic Engineering Targets.

Identification of metabolic engineering targets is a fundamental challenge in strain development programs. While high-throughput (HTP) genetic engineering methodologies capable of generating vast diversity are being developed at a rapid rate, a majority of industrially interesting molecules cannot be screened at sufficient throughput to leverage these techniques. We propose a workflow that couples HTP screening of common precursors (e.g., amino acids) that can be screened either directly or by artificial biosensors, with low-throughput targeted validation of the molecule of interest to uncover nonintuitive beneficial metabolic engineering targets and combinations hereof. Using this workflow, we identified several nonobvious novel targets for improving p-coumaric acid (p-CA) and l-DOPA production from two large 4k gRNA libraries each deregulating 1000 metabolic genes in the yeast Saccharomyces cerevisiae. We initially screened yeast cells transformed with gRNA library plasmids for individual regulatory targets improving the production of l-tyrosine-derived betaxanthins, identifying 30 targets that increased intracellular betaxanthin content 3.5-5.7 fold. Hereafter, we screened the targets individually in a high-producing p-CA strain, narrowing down the targets to six that increased the secreted titer by up to 15%. To investigate whether any of the six targets could be additively combined to improve p-CA production further, we created a gRNA multiplexing library and subjected it to our proposed coupled workflow. The combination of regulating PYC1 and NTH2 simultaneously resulted in the highest (threefold) improvement of the betaxanthin content, and an additive trend was also observed in the p-CA strain. Lastly, we tested the initial 30 targets in a l-DOPA producing strain, identifying 10 targets that increased the secreted titer by up to 89%, further validating our screening by proxy workflow. This coupled approach is useful for strain development in the absence of direct HTP screening assays for products of interest.

Time-resolved transcriptomic and proteomic profiling of Heyndrickxia coagulans during NaOH-buffered L-lactic acid production.

The L-lactic acid (L-LA) fermentation process, based on sodium hydroxide neutralization, demonstrates environmental friendliness during product extraction. However, lactate fermentation is hindered by the pronounced stress effect of sodium lactate on the strain compared with calcium lactate. In this study, we performed time-resolved transcriptomic and proteomic analyses of Heyndrickxia coagulans DSM1 during NaOH-buffered L-LA production. The expression levels of the glycolytic genes demonstrated an initial increase followed by a subsequent decrease, whereas the tricarboxylic acid cycle genes exhibited an initial decrease followed by a subsequent increase throughout the fermentation process. Moreover, we identified clusters of genes consisting of transcription factors and ATP-binding cassette (ABC) transporters that demonstrate a progressive elevation of expression levels throughout the fermentation process, with significant upregulation observed at later stages. This investigation yields valuable insights into the response mechanisms of H. coagulans during NaOH-buffered L-LA fermentation and presents potential targets for metabolic engineering.

Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content

BackgroundMicroalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid; however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be augmented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosynthetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage.ResultsHere, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantitative proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhibited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C. reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photoprotection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further characterization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective proteins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity, and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance.ConclusionsWe used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins (among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency. Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photoprotective mechanisms and the development of a potent microalgal lutein production system.

Discovery of isoflavone phytoalexins in wheat reveals an alternative route to isoflavonoid biosynthesis.

Isoflavones are a group of phenolic compounds mostly restricted to plants of the legume family, where they mediate important interactions with plant-associated microbes, including in defense from pathogens and in nodulation. Their well-studied health promoting attributes have made them a prime target for metabolic engineering, both for bioproduction of isoflavones as high-value molecules, and in biofortification of food crops. A key gene in their biosynthesis, isoflavone synthase, was identified in legumes over two decades ago, but little is known about formation of isoflavones outside of this family. Here we identify a specialized wheat-specific isoflavone synthase, TaCYP71F53, which catalyzes a different reaction from the leguminous isoflavone synthases, thus revealing an alternative path to isoflavonoid biosynthesis and providing a non-transgenic route for engineering isoflavone production in wheat. TaCYP71F53 forms part of a biosynthetic gene cluster that produces a naringenin-derived O-methylated isoflavone, 5-hydroxy-2',4',7-trimethoxyisoflavone, triticein. Pathogen-induced production and in vitro antimicrobial activity of triticein suggest a defense-related role for this molecule in wheat. Genomic and metabolic analyses of wheat ancestral grasses further show that the triticein gene cluster was introduced into domesticated emmer wheat through natural hybridization ~9000 years ago, and encodes a pathogen-responsive metabolic pathway that is conserved in modern bread wheat varieties.

Promiscuous Artemisia annua glycosylphosphatidylinositol-anchored lipid transfer protein 1 (AaLTPG1) promotes terpenoids accumulation in planta

LTPG, encoded by a glycosylphosphatidylinositol-anchored lipid transfer protein family gene, plays a role in exporting fatty acids to the plant surface through the hydrophilic cell wall layer. While soluble extracellular LTPs are known to function in terpenoid accumulation in trichomes, whether membrane-integrated LTPG influences terpenoid accumulation and its underlying mechanisms remains poorly understood. In this study, we report an Artemisia annua LTPG1, which is strongly expressed in floral tissues and positively correlated with three artemisinin biosynthetic genes. The recombinant AaLTPG1 displayed promiscuous binding activity with fatty acids and artemisinin precursors in vitro, resulting in an increase in terpenoids and lipids contents in both AaLTPG1 transgenic tobacco without altering trichome initiation, development, or secretion. Transcriptome sequencing further revealed that ethylene responsive genes were repressed in AaLTPG1-overexpressing tobacco plants. Consistently, artemisinin and its precursors were found to increase transiently in A. annua leaves, and the expression level of ethylene-responsive factors (ERFs) was down-regulated in A. annua. Collectively, this work illustrates the positive regulatory role of LTPG in terpenoids and lipids accumulation by exporting hydrophobic metabolites and repressing ethylene signaling, making AaLTPG1 a potential ideal target for metabolic engineering to improve artemisinin accumulation.

Sustainable production of photosynthetic isobutanol and 3-methyl-1-butanol in the cyanobacterium Synechocystis sp. PCC 6803

BackgroundCyanobacteria are emerging as green cell factories for sustainable biofuel and chemical production, due to their photosynthetic ability to use solar energy, carbon dioxide and water in a direct process. The model cyanobacterial strain Synechocystis sp. PCC 6803 has been engineered for the isobutanol and 3-methyl-1-butanol production by introducing a synthetic 2-keto acid pathway. However, the achieved productions still remained low. In the present study, diverse metabolic engineering strategies were implemented in Synechocystis sp. PCC 6803 for further enhanced photosynthetic isobutanol and 3-methyl-1-butanol production.ResultsLong-term cultivation was performed on two selected strains resulting in maximum cumulative isobutanol and 3-methyl-1-butanol titers of 1247 mg L−1 and 389 mg L−1, on day 58 and day 48, respectively. Novel Synechocystis strain integrated with a native 2-keto acid pathway was generated and showed a production of 98 mg isobutanol L−1 in short-term screening experiments. Enhanced isobutanol and 3-methyl-1-butanol production was observed when increasing the kivdS286T copy number from three to four. Isobutanol and 3-methyl-1-butanol production was effectively improved when overexpressing selected genes of the central carbon metabolism. Identified genes are potential metabolic engineering targets to further enhance productivity of pyruvate-derived bioproducts in cyanobacteria.ConclusionsEnhanced isobutanol and 3-methyl-1-butanol production was successfully achieved in Synechocystis sp. PCC 6803 strains through diverse metabolic engineering strategies. The maximum cumulative isobutanol and 3-methyl-1-butanol titers, 1247 mg L−1 and 389 mg L−1, respectively, represent the current highest value reported. The significantly enhanced isobutanol and 3-methyl-1-butanol production in this study further pave the way for an industrial application of photosynthetic cyanobacteria-based biofuel and chemical synthesis from CO2.