Metabolic Engineering Strategies Research Articles

A Pseudomonas taiwanensis malonyl-CoA platform strain for polyketide synthesis

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L−1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas.

Gibberellic acid overproduction in Fusarium fujikuroi using regulatory modification and transcription analysis.

Gibberellic acid (GA3), one of the natural diterpenoids produced by Fusarium fujikuroi, serves as an important phytohormone in agriculture for promoting plant growth. Presently, the metabolic engineering strategies for increasing the production of GA3 are progressing slowly, which seriously restricted the advancing of the cost-effective industrial production of GA3. In this study, an industrial strain with high-yield GA3 of F. fujikuroi was constructed by metabolic modification, coupling with transcriptome analysis and promoter engineering. The over-expression of AreA and Lae1, two positive factors in the regulatory network, generated an initial producing strain with GA3 production of 2.78g L-1. Compared with a large abundance of transcript enrichments in the GA3 synthetic gene cluster discovered by the comparative transcriptome analysis, geranylgeranyl pyrophosphate synthase 2 (Ggs2), and cytochrome P450-3 genes, two key genes that respectively participated in the initial and final step of biosynthesis, were identified to be downregulated when the highest GA3 productivity was obtained. Employing with a nitrogen-responsive bidirectional promoter, the two rate-limiting genes were dynamically upregulated, and therefore, the production of GA3 was increased to 3.02g L-1. Furthermore, the top 20 upregulated genes were characterized in GA3 over-production, and their distributions in chromosomes suggested potential genomic regions with a high transcriptional level for further strain development. The construction of a GA3 high-yield-producing strain was successfully achieved, and insights into the enriched functional transcripts provided novel strain development targets of F. fujikuroi, offering an efficient microbial development platform for industrial GA3 production. KEY POINTS: • Global regulatory modification was achieved in F. fujikuroi for GA3 overproduction. • Comparative transcriptome analysis revealed bottlenecks in GA specific-pathway. • A dynamically nitrogen-regulated bidirectional promoter was cloned and employed.

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

BackgroundFuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms.ResultsIn this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP.ConclusionsThe results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

Metabolic flux enhancement from the translational fusion of terpene synthases is linked to terpene synthase accumulation

The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), corresponding with increases of a similar magnitude in terpene synthase levels. Our data demonstrate that increased in vivo enzyme levels – resulting from improved expression and/or improved protein stability – is a major driver of catalytic enhancement from enzyme fusion.

Improving the Biodiesel Production in Saccharomyces Cerevisiae: Metabolic Engineering and Enzyme Engineering

The yeast Saccharomyces cerevisiae can produce fuels such as biodiesel using the biomass of lignocellulose. S. cerevisiae metabolize monosaccharide of lignocellulosic hydrolysates to supply fuels/energy globally without competition issue with food supply. Ethanol and free fatty acids (FFAs) are the precursors of biodiesel. S. cerevisiae is able to produce these precursors. S. cerevisiae synthesizes biodiesel from ethanol and fatty acyl-coenzyme A thioesters using wax ester acyltransferase (WS). Here, we review some recent strategies applied to metabolic engineering and enzyme engineering of S. cerevisiae for improving the yield of biodiesel. On the metabolic engineering strategy can be done by directing the carbon flux towards the fatty acids biosynthesis pathway through engineering some genes in the central carbon metabolism. By combined overexpression genes of Acetyl-CoA synthetase (ACS), Acetyl-CoA carboxylase (ACC1), and Fatty Acid Synthase (FAS) will improve biodiesel synthesis. In enzyme engineering, we can remove the allosteric inhibition control of Snf1 to Acetyl-CoA carboxylase ((ACC1), an enzyme that convert Acetyl Co-A to malonyl Co-A) by mutation of two phosphorylation site of ACC1 via site-directed mutagenesis which mutates serine1157,659 to alanine1157,629. This result in enhanced carbon flux to fatty acid synthesis and contribute to a higher yield of biodiesel.

Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis

l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop Bacillus licheniformis as an efficient l-alanine microbial cell factory capable of realizing high-temperature fermentation. By enhancing the glycolytic pathway, knocking out the by-product pathways andoverexpressing the thermostable alanine dehydrogenase, the engineered B. licheniformis strain BLA3 produced 93.7g/L optically pure l-alanine at 50°C. Subsequently, d-alanine dependence of an alanine racemase-deficient strain is relieved by adaptive laboratory evolution, implying that a dormant alternative pathway for d-alanine synthesis is activated in the evolved strain. The d-amino acid aminotransferase Dat1 is shown to be a key enzyme in the dormant alternative pathway. Molecular mechanism of the d-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high-temperature l-alanine production and shows that activating dormant metabolic pathway(s) is an effective strategy of metabolic engineering.

Highlighting the potential of Synechococcus elongatus PCC 7942 as platform to produce α-linolenic acid through an updated genome-scale metabolic modeling.

Cyanobacteria are prokaryotic organisms that capture energy from sunlight using oxygenic photosynthesis and transform CO2 into products of interest such as fatty acids. Synechococcus elongatus PCC 7942 is a model cyanobacterium efficiently engineered to accumulate high levels of omega-3 fatty acids. However, its exploitation as a microbial cell factory requires a better knowledge of its metabolism, which can be approached by using systems biology tools. To fulfill this objective, we worked out an updated, more comprehensive, and functional genome-scale model of this freshwater cyanobacterium, which was termed iMS837. The model includes 837 genes, 887 reactions, and 801 metabolites. When compared with previous models of S. elongatus PCC 7942, iMS837 is more complete in key physiological and biotechnologically relevant metabolic hubs, such as fatty acid biosynthesis, oxidative phosphorylation, photosynthesis, and transport, among others. iMS837 shows high accuracy when predicting growth performance and gene essentiality. The validated model was further used as a test-bed for the assessment of suitable metabolic engineering strategies, yielding superior production of non-native omega-3 fatty acids such as α-linolenic acid (ALA). As previously reported, the computational analysis demonstrated that fabF overexpression is a feasible metabolic target to increase ALA production, whereas deletion and overexpression of fabH cannot be used for this purpose. Flux scanning based on enforced objective flux, a strain-design algorithm, allowed us to identify not only previously known gene overexpression targets that improve fatty acid synthesis, such as Acetyl-CoA carboxylase and β-ketoacyl-ACP synthase I, but also novel potential targets that might lead to higher ALA yields. Systematic sampling of the metabolic space contained in iMS837 identified a set of ten additional knockout metabolic targets that resulted in higher ALA productions. In silico simulations under photomixotrophic conditions with acetate or glucose as a carbon source boosted ALA production levels, indicating that photomixotrophic nutritional regimens could be potentially exploited in vivo to improve fatty acid production in cyanobacteria. Overall, we show that iMS837 is a powerful computational platform that proposes new metabolic engineering strategies to produce biotechnologically relevant compounds, using S. elongatus PCC 7942 as non-conventional microbial cell factory.

Bioengineering for the Microbial Degradation of Petroleum Hydrocarbon Contaminants

Petroleum hydrocarbons are relatively recalcitrant compounds, and as contaminants, they are one of the most serious environmental problems. n-Alkanes are important constituents of petroleum hydrocarbons. Advances in synthetic biology and metabolic engineering strategies have made n-alkane biodegradation more designable and maneuverable for solving environmental pollution problems. In the microbial degradation of n-alkanes, more and more degradation pathways, related genes, microbes, and alkane hydroxylases have been discovered, which provide a theoretical basis for the further construction of degrading strains and microbial communities. In this review, the current advances in the microbial degradation of n-alkanes under aerobic condition are summarized in four aspects, including the biodegradation pathways and related genes, alkane hydroxylases, engineered microbial chassis, and microbial community. Especially, the microbial communities of "Alkane-degrader and Alkane-degrader" and "Alkane-degrader and Helper" provide new ideas for the degradation of petroleum hydrocarbons. Surfactant producers and nitrogen providers as a "Helper" are discussed in depth. This review will be helpful to further achieve bioremediation of oil-polluted environments rapidly.

Biosynthesis of anticancer phytochemical compounds and their chemistry.

Cancer is a severe health issue, and cancer cases are rising yearly. New anticancer drugs have been developed as our understanding of the molecular mechanisms behind diverse solid tumors, and metastatic malignancies have increased. Plant-derived phytochemical compounds target different oncogenes, tumor suppressor genes, protein channels, immune cells, protein channels, and pumps, which have attracted much attention for treating cancer in preclinical studies. Despite the anticancer capabilities of these phytochemical compounds, systemic toxicity, medication resistance, and limited absorption remain more significant obstacles in clinical trials. Therefore, drug combinations of new phytochemical compounds, phytonanomedicine, semi-synthetic, and synthetic analogs should be considered to supplement the existing cancer therapies. It is also crucial to consider different strategies for increased production of phytochemical bioactive substances. The primary goal of this review is to highlight several bioactive anticancer phytochemical compounds found in plants, preclinical research, their synthetic and semi-synthetic analogs, and clinical trials. Additionally, biotechnological and metabolic engineering strategies are explored to enhance the production of bioactive phytochemical compounds. Ligands and their interactions with their putative targets are also explored through molecular docking studies. Therefore, emphasis is given to gathering comprehensive data regarding modern biotechnology, metabolic engineering, molecular biology, and in silico tools.

High-Level Production of Patchoulol in Yarrowia lipolytica via Systematic Engineering Strategies.

Patchoulol is an important sesquiterpene alcohol with a strong and lasting odor, which has led to prominent applications in perfumes and cosmetics. In this study, systematic metabolic engineering strategies were adopted to create an efficient yeast cell factory for patchoulol overproduction. First, a baseline strain was constructed by selecting a highly active patchoulol synthase. Subsequently, the mevalonate precursor pool was expanded to boost patchoulol synthesis. Moreover, a method for downregulating squalene synthesis based on Cu2+-repressible promoter was optimized, which significantly improved the patchoulol titer by 100.9% to 124 mg/L. In addition, a protein fusion strategy resulted in a final titer of 235 mg/L in shake flasks. Finally, 2.864 g/L patchoulol could be produced in a 5 L bioreactor, representing a remarkable 1684-fold increase compared to the baseline strain. To our knowledge, this is the highest patchoulol titer reported so far.

Recent advances in process improvement of dark fermentative hydrogen production through metabolic engineering strategies

Biological means of hydrogen (H2) production has attracted tremendous research and development attention. Dark fermentation provides a possible way of producing H2 from a range of renewable energy sources, including wastewater. During fermentation, various metabolites are formed to create a complex metabolic flux network. Insufficient focus has been placed on the metabolic engineering that is intrinsic to fermentation. This current review summarizes the biochemical pathways occurring in the metabolic network of dark fermentation and how the key operational factors influence metabolism during dark fermentation. Recent developments and strategies for metabolic engineering that have been described to enhance H2 production are recommended. Finally, the economic analysis related to bio-H2 production and prospects is examined. It is envisaged that this study can give beneficial aspects in terms of fundamental knowledge, understanding, and the latest technology for scientists and research engineers in the field of bio-based H2 generation.

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies.

Terpenoids are a diverse group of compounds with isoprene units as basic building blocks. They are widely used in the food, feed, pharmaceutical, and cosmetic industries due to their diverse biological functions such as antioxidant, anticancer, and immune enhancement. With an increase in understanding the biosynthetic pathways of terpenoids and advances in synthetic biology techniques, microbial cell factories have been built for the heterologous production of terpenoids, with the oleaginous yeast Yarrowia lipolytica emerging as an outstanding chassis. In this paper, recent progress in the development of Y.lipolytica cell factories for terpenoid production with a focus on the advances in novel synbio tools and metabolic engineering strategies toward enhanced terpenoid biosynthesis is reviewed.

Engineering the plant metabolic system by exploiting metabolic regulation.

Plants are the most sophisticated biofactories and sources of food and biofuels present in nature. By engineering plant metabolism, the production of desired compounds can be increased and the nutritional or commercial value of the plant species can be improved. However, this can be challenging because of the complexity of the regulation of multiple genes and the involvement of different protein interactions. To improve metabolic engineering (ME) capabilities, different tools and strategies for rerouting the metabolic pathways have been developed, including genome editing and transcriptional regulation approaches. In addition, cutting-edge technologies have provided new methods for understanding uncharacterized biosynthetic pathways, protein degradation mechanisms, protein-protein interactions, or allosteric feedback, enabling the design of novel ME approaches.

Metabolic engineering for the biosynthesis of bis-indolylquinone terrequinone A in Escherichia coli from L-tryptophan and prenol

BackgroundTerrequinone A is a bis-indolylquinone natural product with antitumor activity. Due to its unique asymmetric quinone core structure and multiple functional groups, biosynthesis is more efficient and environmentally friendly than traditional chemical synthesis. Currently, most bis-indolylquinones are obtained by direct extraction from fungi or by chemical synthesis. By focusing on the biosynthesis of terrequinone A, we hope to explore the way to synthesize bis-indolylquinones de novo using Escherichia coli as a cell factory.ResultsIn this study, a terrequinone A synthesis pathway containing the tdiA–tdiE genes was constructed into Escherichia coli and activated by a phosphopantetheinyl transferase gene sfp, enabling the strain to synthesize 1.54 mg/L of terrequinone A. Subsequently, a two-step isopentenol utilization pathway was introduced to enhance the supply of endogenous dimethylallyl diphosphate (DMAPP) in E. coli, increasing the level of terrequinone A to 20.1 mg/L. By adjusting the L-tryptophan (L-Trp)/prenol ratio, the major product could be changed from ochrindole D to terrequinone A, and the content of terrequinone A reached the highest 106.3 mg/L under the optimized culture conditions. Metabolic analysis of L-Trp indicated that the conversion of large amounts of L-Trp to indole was an important factor preventing the further improvement of terrequinone A yield.ConclusionsA comprehensive approach was adopted and terrequinone A was successfully synthesized from low-cost L-Trp and prenol in E. coli. This study provides a metabolic engineering strategy for the efficient synthesis of terrequinone A and other similar bis-indolylquinones with asymmetric quinone cores. In addition, this is the first report on the de novo biosyhthesis of terrequinone A in an engineered strain.

Engineering of a probiotic yeast for the production and secretion of medium-chain fatty acids antagonistic to an opportunistic pathogen Candida albicans.

Candida albicans is an opportunistic pathogen, with its infection as one of the causes of morbidity or mortality. Notably, the probiotic yeast Saccharomyces cerevisiae var. boulardii has shown the potential to fight against Candida infections. In this study, we aimed to engineer a commercial boulardii strain to produce medium-chain fatty acids (MCFAs) with antagonistic effects against C. albicans. First, we identified and characterized a boulardii strain and created its auxotrophic strain Δura3. Next, we constructed and expressed a heterologous MCFA biosynthetic pathway under the control of inducible and constitutive promoters. Aside from examining MCFA production and secretion, we confirmed MCFAs' effects on C. albicans' anti-biofilm and anti-hyphal formations and the immunomodulatory effect of MCFA-containing supernatants on Caco-2 cells. We found that under constitutive promoters, the engineered boulardii strain constitutively produced and secreted a mixture of C6:0, C8:0, and C10:0. The secreted MCFAs then reduced biofilm and hyphal formations in C. albicans SC5314. We also confirmed that MCFAs upregulated the expression of virulence-related genes in SC5314. Furthermore, we found that the constitutively produced MCFAs in the supernatant induced the upregulation of immune response genes in Caco-2 cells co-cultured with SC5314, indicating MCFAs' roles in immunomodulation. Overall, the engineered boulardii strain produced and secreted MCFAs, as well as demonstrated antagonistic effects against C. albicans SC5314 and immune-modulatory effects in Caco-2. To our knowledge, this represents the first study tackling the metabolic engineering of a commercial probiotic yeast strain to constitutively produce and secrete MCFAs showing anti-Candida effects. Our study forms the basis of the potential development of a live biotherapeutics probiotic yeast against Candida infections through metabolic engineering strategies.

Systems metabolic engineering of Escherichia coli for hyper-production of 5‑aminolevulinic acid

Background5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization.ResultsIn this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction.ConclusionsAn industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.

Bioconversion of methanol to 3-hydroxypropionate by engineering Ogataea polymorpha

Methanol bioconversion toward chemical production can be helpful for carbon neutrality. Here metabolic engineering an industrial methylotrophic yeast Ogataea polymorpha was conducted for overproduction of 3-hydroxypropionate (3-HP), an important platform chemical, which can be used for production of special chemicals including acrylamide, acrylic acid, 1,3-propanediol, as well as biodegradable plastics. We developed several metabolic engineering strategies to optimize the 3-HP biosynthetic pathway and enhance the supply of precursors acetyl-CoA and malonyl-CoA, and cofactor NADPH, which enabled the 3-HP production of 1.45 g/L under batch fermentation and 7.10 g/L under fed-batch fermentation at shake flask scale, with a yield of 0.14 g/g methanol. This was, to our knowledge, the highest 3-HP titer from methanol and even one-carbon sources. This study demonstrated the potential of O. polymorpha as a cell factory for chemical production from methanol.

Compartmentalization engineering of yeasts to overcome precursor limitations and cytotoxicity in terpenoid production.

Metabolic engineering strategies for terpenoid production have mainly focused on bottlenecks in the supply of precursor molecules and cytotoxicity to terpenoids. In recent years, the strategies involving compartmentalization in eukaryotic cells has rapidly developed and have provided several advantages in the supply of precursors, cofactors and a suitable physiochemical environment for product storage. In this review, we provide a comprehensive analysis of organelle compartmentalization for terpenoid production, which can guide the rewiring of subcellular metabolism to make full use of precursors, reduce metabolite toxicity, as well as provide suitable storage capacity and environment. Additionally, the strategies that can enhance the efficiency of a relocated pathway by increasing the number and size of organelles, expanding the cell membrane and targeting metabolic pathways in several organelles are also discussed. Finally, the challenges and future perspectives of this approach for the terpenoid biosynthesis are also discussed.

EtiBsu1209: A comprehensive multiscale metabolic model for Bacillus subtilis.

Genome-scale metabolic models (GEMs) have been widely used to guide the computational design of microbial cell factories, and to date, seven GEMs have been reported for Bacillus subtilis, a model gram-positive microorganism widely used in bioproduction of functional nutraceuticals and food ingredients. However, none of them are widely used because they often lead to erroneous predictions due to their low predictive power and lack of information on regulatory mechanisms. In this work, we constructed a new version of GEM for B. subtilis (iBsu1209), which contains 1209 genes, 1595 metabolites, and 1948 reactions. We applied machine learning to fill gaps, which formed a relatively complete metabolic network able to predict with high accuracy (89.3%) the growth of 1209 mutants under 12 different culture conditions. In addition, we developed a visualization and code-free software, Model Tool, for multiconstraints model reconstruction and analysis. We used this software to construct etiBsu1209, a multiscale model that integrates enzymatic constraints, thermodynamic constraints, and transcriptional regulatory networks. Furthermore, we used etiBsu1209 to guide a metabolic engineering strategy (knocking out fabI and yfkN genes) for the overproduction of nutraceutical menaquinone-7, and the titer increased to 153.94 mg/L, 2.2-times that of the parental strain. To the best of our knowledge, etiBsu1209 is the first comprehensive multiscale model for B. subtilis and can serve as a solid basis for rational computational design of B. subtilis cell factories for bioproduction.

Transcriptome analysis of transcription factors and enzymes involved in monoterpenoid biosynthesis in different chemotypes of Mentha haplocalyx Briq.

The main active ingredients of Mentha haplocalyx Briq. essential oils are monoterpenes. According to the component of essential oils, M. haplocalyx can be divided into different chemotypes. Chemotype variation is widespread in Mentha plants but its formation mechanism is unclear. We selected the stable chemotype l-menthol, pulegone, and carvone of M. haplocalyx for transcriptome sequencing. To further investigate the variation of chemotypes, we analyzed the correlation between differential transcription factors (TFs) and key enzymes. Fourteen unigenes related to monoterpenoid biosynthesis were identified, among which (+)-pulegone reductase (PR) and (-)-menthol dehydrogenase (MD) were significantly upregulated in l-menthol chemotype and (-)-limonene 6-hydroxylase was significantly upregulated in carvone chemotype. In addition, 2,599 TFs from 66 families were identified from transcriptome data and the differential TFs included 113 TFs from 34 families. The families of bHLH, bZIP, AP2/ERF, MYB, and WRKY were highly correlated with the key enzymes PR, MD, and (-)-limonene 3-hydroxylase (L3OH) in different M. haplocalyx chemotypes (r > 0.85). The results indicate that these TFs regulate the variation of different chemotypes by regulating the expression patterns of PR, MD, and L3OH. The results of this study provide a basis for revealing the molecular mechanism of the formation of different chemotypes and offer strategies for effective breeding and metabolic engineering of different chemotypes in M. haplocalyx.