Metabolic Engineering Strategies Research Articles

Metabolic Engineering for Squalene Production: Advances and Perspectives.

Squalene is a linear polyunsaturated triterpene which has multiple physiological functions including anticancer, antioxidant, and skin-care. It has been widely used in the food, medicine, and cosmetics sectors and also serves as a precursor of triterpenes and steroids. Recently, the production of squalene by microbial cell hosts has drawn much attention due to its sustainability, environmental friendliness, and great efficiency. In this review, we first introduce the recent developments in the production of squalene by employing microbial cell factories, especially yeasts. Next, we underscore the primary metabolic strategies, including the biosynthetic pathway engineering, precursor manipulation, cofactor engineering, and organelle engineering. In addition to traditional metabolic engineering strategies, we also discuss some prospective metabolic regulation approaches, including regulation of lipid synthesis, identifying and manipulating related transcription factors, dynamic regulation of the metabolic pathway, and secretion engineering of membrane-impermeable terpenoids. These approaches provide insights for further metabolic engineering of squalene and related terpenoids.

Biotechnology Advances in Natural Food Colorant Acylated Anthocyanin Production

ABSTRACTThe food application of natural blue colorants such as acylated anthocyanin is becoming increasingly popular due to their safety, nutritional value, organoleptic attributes, and more pH/thermo‐stable properties compared to nonacylated anthocyanins. However, it is a great challenge to obtain enough acylated anthocyanins to meet market demand, rather than readily available in nature from edible fruits and vegetables. In this review, the acylated anthocyanins in food crops, the efficient and eco‐friendly extraction of food‐grade acylated anthocyanins, and their potential application in intelligent packaging are extensively summarized. Subsequently, the plant anthocyanin acyltransferases (AATs) are also comprehensively summarized. The strategy of functional optimization of AATs through computational or artificial intelligence–based protein‐directed evolution is described and suggested. Additionally, the advantages and disadvantages of acylated anthocyanins generated in microbes and plant suspension cells are extensively compared, and the chassis selection of engineering acylated anthocyanins is discussed. Finally, the approaches and strategies of metabolic engineering to enhance the yield of acylated anthocyanins in plant suspension cells are also described and discussed. Taken together, this review provides a roadmap or strategy for the production of acylated anthocyanins to meet the increasing market demand in future food industry.

Functional Profiling of Abundant Glycosyltransferases in the Rhizospheric Bacteriome of Abutilon fruticosum

In this study, we undertook an extensive exploration of the rhizospheric bacteriome of Abutilon fruticosum, focusing on the activities of Carbohydrate-Active Enzymes (CAZymes) of class glycosyltransferases (GTs), and their potential impact on plant growth and fortification against environmental adversities and soil-borne pathogens. Investigation revealed a predominance of families GT1, GT31, GT35, GT39, and GT51, mainly ascribed to bacterial genera Microvirga, Arthrobacter, Blastococcus, Bacillus, Nocardioides, and Kocuria. Our findings unveiled the pivotal role of phenylalanine, mostly exuded by plant roots, serving as the primary substrate for the catalytic activities of the identified CAZymes. With glucosylation emerging as the predominant enzymatic function, we propose UDP-D-glucose as the central core intermediate metabolite, orchestrating a multifaceted network of interrelated metabolic pathways. Subsequent profiling delineated a diverse array of metabolites synthesized by 10 CAZymes actively participating in 10 crosstalking pathways. Functional analyses of these metabolites underscore their significance in the biosynthesis of flavonoids, anthocyanins, and related derivatives, crucial for the reproductive success of plants by serving as visual cues for insect pollinators and conferring protection against deleterious effects of radiation. Certain other metabolites play vital roles in reinforcing the structural integrity of plant cell walls, thus enhancing resistance against enzymatic degradation while augmenting cytokinin activity. In conclusion, our study elucidates a novel paradigm of plant-bacteria mutualistic interaction, offering insights into the intricate mechanisms underlying plant-microbe symbiosis. We posit that the integration of high-throughput technologies with metabolic engineering strategies presents a promising approach for applying cost-effective and environmentally benign alternatives to conventional agricultural practices. (249 words)

Increased accumulation of fatty acids in engineered Saccharomyces cerevisiae by co-overexpression of interorganelle tethering protein and lipases

Fatty acids (FAs) and their derivatives are versatile chemicals widely used in various industries. Synthetic biology, using microbial cell factories, emerges as a promising alternative technology for FA production. To enhance the production capacity of these microbial chassis, additional engineering strategies are imperative. Based on the comparison of the morphological changes of lipid droplets (LDs) between oleaginous and non-oleaginous yeasts, we developed a new engineering strategy to increase the accumulation of FAs in Saccharomyces cerevisiae through manipulation of regulation factor and lipases related to LD. The increased biogenesis of LDs, achieved by overexpressing the interorganelle tethering protein Mdm1, coupled with the accelerated degradation of LDs through upregulated lipases, resulted in a 10.70-fold increase in total FAs production. Co-overexpression of Mdm1 and selected lipases significantly improved the biosynthesis of FAs and linoleic acid in the engineered S. cerevisiae. The efficient LD-based metabolic engineering strategy presented in this study holds the potential to advance the high-level production of FAs and their derivatives in microbial cell factories.

Semi-rational design and modification of phosphoketolase to improve the yield of tyrosol in Saccharomyces cerevisiae

Tyrosol is an important component of pharmaceuticals, nutraceuticals, and cosmetics, and their biosynthetic pathways are currently a hot research topic. d-Erythrose 4-phosphate is a key precursor for the biosynthesis of tyrosol in Saccharomyces cerevisiae. Hence, the flux of d-Erythrose 4-phosphate determined the yield of tyrosol synthesis. In this study, we first obtained an S. cerevisiae strain S19 with a tyrosol yield of 247.66 mg/L by metabolic engineering strategy. To increase the production of d-Erythrose 4-phosphate, highly active phosphoketolase BA-C was obtained by bioinformatics combined with tyrosol yield assay. The key residue sites 183, 217, and 320 were obtained by molecular docking, kinetic simulation, and tyrosol yield verification. After mutation, the highly efficient phosphoketolase BA-CHis320Met was obtained, with a 37.32 % increase in enzyme activity. The tyrosol production of strain S26 with BA-CHis320Arg increased by 43.05 % than strain S25 with BA-C and increased by 151.19 % compared with the strain S19 without phosphoketolase in a 20 L fermenter. The mining and modification of phosphoketolase will provide strong support for the de novo synthesis of aromatic compounds.

Efficient Production of 3'-Sialyllactose Using Escherichia coli.

3'-Sialyllactose (3'-SL), a key component of human milk oligosaccharides, provides significant health benefits and immune modulation, and is increasingly used in infant formula and dietary supplements. This study presents a novel approach for the efficient biosynthesis of 3'-SL using Escherichia coli BL21star(DE3)ΔlacZ through genomic integration. We first addressed the issue of metabolic competition by deleting crucial genes, nanA, nanK, nanE, and nanT, that are involved in the degradation of N-acetylneuraminic acid. This strategic gene knockout minimized the flux through competing pathways. The engineered Escherichia coli strain was subsequently transformed with the exogenous genes neuBCA and nST, enabling the de novo synthesis of 3'-SL. A modular metabolic engineering strategy was utilized to optimize the expression of key enzymes within the MSU module, enhancing and balancing the carbon flux distribution. Additionally, a cofactor regeneration strategy was implemented to increase CTP availability, which improved cofactor recycling and fine-tuned the metabolic pathway for maximal 3'-SL production. Transport protein screening was incorporated to further increase the extracellular concentration of 3'-SL, resulting in an unprecedented yield of 56.8 g/L in a 5L bioreactor fermentation, setting a new benchmark in the field.

Optimized Production of Concanamycins Using a Rational Metabolic Engineering Strategy

Plecomacrolides, such as concanamycin and bafilomycin, are potent and specific inhibitors of vacuolar-type ATPase. Concanamycins are 18-membered macrolides with promising therapeutic potential against multiple diseases, including viral infection, osteoporosis, and cancer. Due to the complexity of their total synthesis, the production of concanamycins is only achieved through microbial fermentation. However, the low titers of concanamycin A and its analogs in the native producing strains are a significant bottleneck for scale-up, robust structure-activity relationship studies, and drug development. To address this challenge, we designed a library of engineered Streptomyces strains for the overproduction of concanamycins by combining the overexpression of target regulatory genes with the optimization of fermentation media. Integration of two endogenous regulators from the concanamycin biosynthetic gene cluster (cms) and one heterologous regulatory gene from the bafilomycin biosynthetic gene cluster into the attB site significantly increased production of concanamycin A and its low abundant analog concanamycin B in Streptomyces eitanensis. The highest titers reported to date were observed in the engineered S. eitanensis DHS10676, which produced over 900 mg/L of concanamycin A and 300 mg/L of concanamycin B. Heterologous overexpression of the identified target regulatory genes across a panel of Streptomyces spp., harboring a putative concanamycin biosynthetic gene cluster confirmed its identity, and significantly improved concanamycin A production in all tested strains. Strain engineering, optimization of fermentation, and extraction purification protocols enabled swift access to these structurally complex plecomacrolides for semi-synthetic medicinal chemistry-based approaches. Together, this work established a platform for robust overproduction of concanamycin analogs across species.

De Novo Biosynthesis of a Polyene-Type Ginsenoside Precursor Dammaradienol in Saccharomyces cerevisiae.

Typical dammarane-type ginsenosides are well-known tetracyclic triterpenoids with significant pharmacological effects including antitumor, cardiovascular protection, and neuroprotection. Polyene-type ginsenosides exhibit stronger biological activities than common ginsenosides; however, their contents are low, and most are converted from ginsenosides through a series of processing steps, resulting in higher preparation costs. In this study, a dammaradienol synthase, AarOSC20433, was identified for the first time from Artemisia argyi H. Lév. & Vaniot (A. argyi). The high-yielding squalene strain constructed in this study was used as the chassis strain. Yeast heterologous biosynthesis of the polyene-type ginsenoside precursor dammaradienol was achieved via metabolic engineering strategies, including optimization of the terpene supply, increase in copy number of AarOSC20433, and rational enzyme design. Eventually, through replenishment and batch fermentation, the titer of dammaradienol reached 1.037 g/L (4.3 mg/L/OD), laying a solid foundation for the construction of a polyene-type ginsenoside cell factory.

Harnessing the optimization of enzyme catalytic rates in engineering of metabolic phenotypes.

The increasing availability of enzyme turnover number measurements from experiments and of turnover number predictions from deep learning models prompts the use of these enzyme parameters in precise metabolic engineering. Yet, there is no computational approach that allows the prediction of metabolic engineering strategies that rely on the modification of turnover numbers. It is also unclear if modifications of turnover numbers without alterations in the host's transcriptional regulatory machinery suffice to increase the production of chemicals of interest. Here, we present a constraint-based modeling approach, termed Overcoming Kinetic rate Obstacles (OKO), that uses enzyme-constrained metabolic models to predict in silico strategies to increase the production of a given chemical, while ensuring specified cell growth. We demonstrate that the application of OKO to enzyme-constrained metabolic models of Escherichia coli and Saccharomyces cerevisiae results in strategies that can at least double the production of over 40 compounds with little penalty to growth. Interestingly, we show that the overproduction of compounds of interest does not entail only an increase in the values of turnover numbers. Lastly, we demonstrate that a refinement of OKO, allowing also for manipulation of enzyme abundance, facilitates the usage of the available compendia and deep learning models of turnover numbers in the design of precise metabolic engineering strategies. Our results expand the usage of genome-scale metabolic models toward the identification of targets for protein engineering, allowing their direct usage in the generation of innovative metabolic engineering designs for various biotechnological applications.

Combinatorial metabolic engineering strategy of precursor pools for the yield improvement of spinosad in Saccharopolyspora spinosa

Spinosad is an insecticide produced by Saccharopolyspora spinosa, and its larvicidal activity is considered a promising approach to combat crop pests. The aim of this study was to enhance the synthesis of spinosad through increasing the supply of acyl-CoAs precursor by the following steps. (i) Engineering the β-oxidation pathway by overexpressing key genes within the pathway to promote the synthesis of spinosad. The results showed that the overexpression of fadD, fadE, and fadA1 genes, as well as the co-expression of fadA1 and fadE genes, increased the yield of spinosad by 0.36-fold, 0.89-fold, 0.75-fold and 1.25-fold respectively. (ii) Employing combinatorial engineering of the β-oxidation pathway and ACC/PCC pathway to promote the synthesis of spinosad. The results showed that the co-expression of fadE and pccA, as well as accC and fadE, resulted in a 1.77-fold and 1.43-fold increase in spinosad production respectively. (iii) When exogenous triacylglycerol was added to the fermentation medium, the solely engineering of the β-oxidation pathway increased the yield of spinosad by 7.13-fold, reaching 427.23 mg/L. While the combinatorial engineering of both the β-oxidation pathway and ACC/PCC pathway increased the yield of spinosad by 9.61-fold, reaching 625.17 mg/L, and further optimization of the culture medium resulted in an even higher yield of spinosad, reaching 1293.43 mg/L. The results of this study indicate that the above combination strategy can promote the efficient biosynthesis of spinosad.

Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics.

Production of L-lactic acid from methanol by engineered yeast Pichia pastoris

Lactic acid (LA) serves as a widely used platform compound and has received significant attention as a raw material for synthesis of biodegradable polylactic acid. Currently, LA is mainly produced through microbial fermentation, but its high costs undermine its competitive advantage against other materials, necessitating the development of novel production routes. Methanol bioconversion represents an emerging low-carbon circular economy, where LA could become an outstanding representative product. This study successfully established an efficient methanol-based LA synthesis route in Pichia pastoris. Through systematic metabolic engineering strategies, including screening lactate dehydrogenase, modification of cofactor preference, blocking LA consumption pathway, and mitochondrial LA synthesis compartmentalization, 4.2 g/L L-LA was produced in fed-batch fermentation by using methanol as the sole carbon source. Through multi-dimensional and spatial engineering of enzyme, a cell factory was developed for efficient synthesis of L-LA, highlights the significant potential of the low-carbon synthesis route for L-LA via methanol bioconversion.

Metabolic engineering of Corynebacterium glutamicum for the production of pyrone and pyridine dicarboxylic acids

Environmental concerns from plastic waste are driving interest in alternative monomers from bio-based sources. Pseudoaromatic dicarboxylic acids are promising alternatives with chemical structures similar to widely used petroleum-based aromatic dicarboxylic acids. However, their use in polyester synthesis has been limited due to production challenges. Here, we report the fermentative production of five pseudoaromatic dicarboxylic acids, including 2-pyrone-4,6-dicarboxylic acid (PDC) and pyridine dicarboxylic acids (PDCAs: 2,3-, 2,4-, 2,5-, and 2,6-PDCA), from glucose using five engineered Corynebacterium glutamicum strains. A platform C. glutamicum chassis strain was constructed by modulating the expression of nine genes involved in the synthesis and degradation pathways of precursor protocatechuate (PCA) and the glucose-uptake system. Comparative transcriptome analysis of the engineered strain against wild-type C. glutamicum identified iolE (NCgl0160) as a target for PDC production. Optimized fed-batch fermentation conditions enabled the final engineered strain to produce 76.17 ± 1.24 g/L of PDC. Using this platform strain, we constructed 2,3-, 2,4-, and 2,5-PDCA-producing strains by modulating the expression of key enzymes. Additionally, we demonstrated a previously uncharacterized pathway for 2,3-PDCA biosynthesis. The engineered strains produced 2.79 ± 0.005 g/L of 2,3-PDCA, 494.26 ± 2.61 mg/L of 2,4-PDCA, and 1.42 ± 0.02 g/L of 2,5-PDCA through fed-batch fermentation. To complete the portfolio, we introduced the 2,6-PDCA biosynthetic pathway to an L-aspartate pathway-enhanced C. glutamicum strain, producing 15.01 ± 0.03 g/L of 2,6-PDCA in fed-batch fermentation. The metabolic engineering strategies developed here will be useful for the production of pseudoaromatic chemicals.

Construction and Optimization of a Yeast Cell Factory for Producing Active Unnatural Ginsenoside 3β-O-Glc2-DM.

Ginsenosides are major active components of Panax ginseng, which are generally glycosylated at C3-OH and/or C20-OH of protopanaxadiol (PPD) and C6-OH and/or C20-OH of protopanaxatriol. However, the glucosides of dammarenediol-II (DM), which is the direct precursor of PPD, have scarcely been separated from P. ginseng. Because different positions and numbers of the hydroxyl and glycosyl groups lead to a diversity of structure and function of the ginsenosides, it can be inferred that DM glucosides may have different pharmacological activities compared with natural ginsenosides. Herein, we first constructed the cell factory for de novo biosynthesis of 3-O-(β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl)-dammar-24-ene-3β,20S-diol (3β-O-Glc2-DM) by introducing the codon-optimized genes encoding dammarenediol-II synthase, two UDP-glycosyltransferases (UGTs) including UGT74AC1-M7 from Siraitia grosvenorii and UGTPg29 from P. ginseng in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titer of 3β-O-Glc2-DM was then increased from 18.9 to 148.0 mg/L by several metabolic engineering strategies including overexpressing the rate-limiting enzymes of triterpenoid biosynthesis, balancing carbon flux of biosynthetic pathways of triterpenoid and ergosterol, and engineering endoplasmic reticulum. Furthermore, the 3β-O-Glc2-DM titer of 766.3 mg/L was achieved through fed-batch fermentation in a 3-L bioreactor. Finally, in vitro assays demonstrated that 3β-O-Glc2-DM exhibited a protective effect on H/R-induced cardiomyocyte damage. This work provides a feasible approach for production of 3β-O-Glc2-DM as a potential cardioprotective drug candidate.

The Functional Characterization of the 6-Phosphogluconate Dehydratase Operon in 2-Ketogluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01.

Genus Pseudomonas bacteria mainly consume glucose through the Entner-Doudoroff (ED) route due to a lack of a functional Embden-Meyerhof-Parnas (EMP) pathway. In the present study, a 6-phosphogluconate dehydratase (edd) operon in the ED route was well investigated to find its structural characteristics and roles in the regulation of glucose consumption and 2-ketogluconic acid (2KGA) metabolism in the industrial 2KGA-producer P. plecoglossicida JUIM01. The edd operon contained four structural genes of edd, glk, gltR, and gtrS, encoding 6-PG dehydratase Edd, glucokinase Glk, response regulatory factor GltR, and histidine kinase GtrS, respectively. A promoter region was observed in the 5'-upstream of the edd gene, with a transcriptional start site located 129 bp upstream of the edd gene and in a pseudo-palindromic sequence of 5'-TTGTN7ACAA-3' specifically binding to the transcription factor HexR. The knockout of the edd gene showed a remarkably negative effect on cell growth and re-growth using 2KGA as a substrate, beneficial to 2KGA production, with an increase of 8%. The deletion of glk had no significant effect on the cell growth or glucose metabolism, while showing an adverse impact on the 2KGA production, with a decrease of 5%. The outputs of the present study would provide a theoretical basis for 2KGA-producer improvement with metabolic engineering strategies and the development and optimization of P. plecoglossicida as the chassis cells.

Proteomic and Targeted Lipidomic Analyses of Fluid and Rigid Rubber Particle Membrane Domains in Guayule.

Rubber (cis-1,4-polyisoprene) is produced in cytosolic unilamellar vesicles called rubber particles (RPs), and the protein complex responsible for this synthesis, the rubber transferase (RTase), is embedded in, or tethered to, the membranes of these RPs. Solubilized enzyme activity is very difficult to achieve because the polymerization of highly hydrophilic substrates into hydrophobic polymers requires a polar/non-polar interface and a hydrophobic compartment. Using guayule (Parthenium argentatum) as a model rubber-producing species, we optimized methods to isolate RP unilamellear membranes and then a subset of membrane microdomains (detergent-resistant membranes) likely to contain protein complexes such as RTase. The phospholipid and sterol composition of these membranes and microdomains were analyzed using thin-layer chromatography (TLC) and liquid chromatography tandem mass spectroscopy (LC-MS/MS). Our data indicate that RP membranes consist predominantly of phosphatidic acid-containing membrane microdomains (DRMs or "lipid rafts"). Proteomic analyses of guayule RP membranes and membrane microdomains identified 80 putative membrane proteins covering 30 functional categories. From this population, we have tentatively identified several proteins in multiple functional domains associated with membrane microdomains which may be critical to RTase function. Definition of the mechanisms underlying rubber synthesis will provide targets for both metabolic engineering and breeding strategies designed to increase natural rubber production in latex-producing species.

Genomic and metabolomic insights into the selection and differentiation of bioactive compounds in citrus

Bioactive compounds play an increasingly prominent role in breeding functional and nutritive fruit crops such as citrus. However, the genomic and metabolic bases for the selection and differentiation underlying bioactive compound variations in citrus remain poorly understood. In this study, we constructed a species-level variation atlas of genomes and metabolomes using 299 citrus accessions. A total of 19 829 significant SNPs were targeted to 653 annotated metabolites, among which multiple significant signals were identified for secondary metabolites, especially flavonoids. Significant differential accumulation of bioactive compounds in the phenylpropane pathway, mainly flavonoids and coumarins, was unveiled across ancestral citrus species during differentiation, which is likely associated with the divergent haplotype distribution and/or expression profiles of relevant genes, including p-coumaroyl coenzyme A 2′-hydroxylases, flavone synthases, cytochrome P450 enzymes, prenyltransferases, and UDP-glycosyltransferases. Moreover, we systematically evaluated the beneficial bioactivities such as the antioxidant and anticancer capacities of 219 citrus varieties, and identified robust associations between distinct bioactivities and specific metabolites. Collectively, these findings provide citrus breeding options for enrichment of beneficial flavonoids and avoidance of potential risk of coumarins. Our study will accelerate the application of genomic and metabolic engineering strategies in developing modern healthy citrus cultivars.

Microbial production of 5-epi-jinkoheremol, a plant-derived antifungal sesquiterpene.

Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from Catharanthus roseus, 5-epi-jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and m6A writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5-epi-jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5-epi-jinkoheremol synthase TPS18, resulting in the variant TPS18I21P/T414S, with the most improved properties. Finally, the titer of 5-epi-jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5-epi-jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5-epi-jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5-epi-jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5-epi-jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.

Promotion of gentiopicroside production and transcriptional responses of biosynthetic genes in adventitious root cultures of Gentiana scabra Bunge by elicitation with methyl jasmonate

Abiotic elicitors play a crucial role in regulating various aspects of plant growth, development, and specialized metabolism. This study aimed to further increase the gentiopicroside content by screening elicitor types, optimizing elicitation conditions, and estimating transcriptional responses of biosynthetic genes in the adventitious roots of Gentiana scabra. The results showed that methyl jasmonate (MeJA) was the most effective inducer for biomass accumulation in the adventitious roots of G. scabra among tested elicitors, with fresh weight (FW) and dry weight (DW) of 13.26 ± 0.57 g flask−1 and 1.31 ± 0.25 g flask−1, respectively. The effects of the induction time and concentration of MeJA on the biomass and gentiopicroside content in the adventitious roots of G. scabra were investigated. The maximum FW (15.73 ± 0.41 g flask−1) and DW (1.51 ± 0.19 g flask−1) were obtained when the roots were cultured for 6 days in MS liquid medium containing 3.0 mg L−1 1-naphthlcetic acid (NAA) and 1.0 mg L−1 kinetin (KT) at MeJA concentration of 100 μM L−1. Also, the gentiopicroside content significantly increased to 62.62 ± 0.27 mg g−1 DW, and was 2.49 times higher than that for the nontreated control. The expression levels of 12 candidate gentiopicroside biosynthesis–related genes involved in the mevalonic acid (MVA), methyl erythritol phosphate (MEP), and secoiridoid pathways were estimated in the adventitious roots of G. scabra to further understand the transcriptional response to MeJA elicitation. Among these, 10 genes (ACCT1, HMGR1, MCK1, MVD1, GPPS4, G10H, IS3, DL7H1, DXS5, and ISPH5) were upregulated whereas DXR1 and IDI1 genes were downregulated in the adventitious roots of G. scabra compared with nontreated control, with significant differences having threshold P value ≤0.05. The transcriptional analyses revealed that 12 candidate genes were the key regulated genes in the gentiopicroside biosynthetic pathway. Overall, the findings provided a promising, feasible, and stable approach to utilizing MeJA elicitation to increase the production of valuable gentiopicroside. Additionally, they provided a foundation for future gentiopicroside biosynthesis through metabolic engineering strategies in the adventitious roots of G. scabra.

Unveiling Metabolic Engineering Strategies by Quantitative Heterologous Pathway Design.

Constructing efficient cell factories requires the rational design of metabolic pathways, yet quantitatively predicting the potential pathway for breaking stoichiometric yield limit in hosts remains challenging. This leaves it uncertain whether the pathway yield of various products can be enhanced to surpass the stoichiometric yield limit and whether common strategies exist. Here, a high-quality cross-species metabolic network model (CSMN) and a quantitative heterologous pathway design algorithm (QHEPath) are developed to address this challenge. Through systematic calculations using CSMN and QHEPath, 12,000 biosynthetic scenarios are evaluated across 300 products and 4 substrates in 5 industrial organisms, revealing that over 70% of product pathway yields can be improved by introducing appropriate heterologous reactions. Thirteen engineering strategies, categorized as carbon-conserving and energy-conserving, are identified, with 5 strategies effective for over 100 products. A user-friendly web server is developed to quantitatively calculate and visualize the product yields and pathways, which successfully predicts biologically plausible strategies validated in literature for multiple products.