Metabolic Engineering Communications

Extensive usage of plant-based oils, especially palm oil, has led to environmental and social issues, such as deforestation and loss of biodiversity, thus sustainable alternatives are required. Microbial oils, especially from Yarrowia lipolytica, offer a promising solution due to their similar composition to palm oil, low carbon footprint, and ability to utilize low-cost substrates. In this study, we employed the Design-Build-Test-Learn (DBTL) approach to enhance lipid production in Y. lipolytica. We systematically evaluated predictions from the genome-scale metabolic model to identify and overcome bottlenecks in lipid biosynthesis. We tested the effect of predicted medium supplements and genetic intervention targets, including the overexpression of ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), threonine synthase (TS), diacylglycerol acyltransferase(DGA1), the deletion of citrate exporter gene (CEX1) and disruption of {beta}-oxidation pathway (MFE1). Combining TS and DGA1 overexpression in the{Delta} mfe_{Delta}cex background achieved a remarkable 200% increase in lipid content (56 % w/w) and a 230% increase in lipid yield on glycerol. These findings underscore the potential of Y. lipolytica as an efficient microbial cell factory for fatty acid production. Our study advances the understanding of lipid metabolism in Y. lipolytica and demonstrates a viable approach for developing sustainable and economically feasible alternatives to palm oil. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/606002v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1394cf6org.highwire.dtl.DTLVardef@ebdd5eorg.highwire.dtl.DTLVardef@1126ab2org.highwire.dtl.DTLVardef@1ae028_HPS_FORMAT_FIGEXP M_FIG C_FIG We followed the Design-Build-Test-Learn approach to identify and overcome bottlenecks in lipid biosynthesis in Y. lipolytica. DBTL intertwined the predictions from the metabolic model with addressed bottlenecks, investigated the effect of genetic interventions and medium supplements on lipid content, and ultimately defined an efficient strain design strategy.

Production of value-added, plant-derived compounds in microbes increasingly attracts commercially interest in food and pharmaceutical industries. However, plant metabolic pathways are complex, require a robust balance of enzymes, cofactors, ATP and other metabolites, and often result in low production when transplanted to bacteria. This is exemplified by the biosynthesis of curcuminoids from the Curcuma longa plant. Here, we combine dynamic pathway modeling, systematic testing of isoenzymes, and the optimization of gene expression levels and substrate concentrations for the biosynthesis of curcuminoids in Pseudomonas putida, leading to unprecedented conversion rates of caffeic acid and tyrosine to curcumin. The development of kinetic ensemble models guided the design of production strains, emphasizing the necessity of high relative expression of c3h, curs2 and dcs and, the low relative expression of tal, comt, ccoaomt, and 4cl4. This optimization resulted in a strain that achieved a 10.8 {+/-}1.8% of the maximum theoretical yield of curcumin from tyrosine. This represents a 4.1-fold increase in production efficiency and the highest yield reported to date, demonstrating the potential of P. putida as a promising platform for curcuminoid production. Our findings highlight the effectiveness of our strategy not only in the advances in the production of curcuminoids but also in setting a framework for the biosynthesis of other complex compounds.

Lactones constitute a family of aroma compounds found in fruits, flowers and vegetables and are in high demand in the food industry. Previous studies have reported biotransformation of castor beans-extracted ricinoleic acid to {gamma}-decalactone using oleaginous yeast Yarrowia lipolytica. Given the potential toxicities associated with castor beans, we have used a metabolic flux-engineering approach to produce {gamma}-decalactone from oleic acid in Saccharomyces cerevisiae. Intracellular conversion of oleic acid to ricinoleic acid was achieved by the expression of oleate hydroxylase Fah12 from ergot fungus Claviceps purpurea. Glycerol-3-phosphate dehydrogenase-mediated glycerol synthesis was identified as the major metabolic diversion of oleic acid that negatively impacts {gamma}-decalactone yields. Chemogenomic profiling analysis revealed that the tryptophan biosynthetic pathway provides resistance to {gamma}-decalactone-mediated toxicity in yeast. Overexpression of tryptophan transporter Tat1 enhanced {gamma}-decalactone production by about 3- fold. Deficiency of genes encoding the cytoplasmic fatty acyl CoA synthetases FAA1 or FAA4 alone did not significantly influence {gamma}-decalactone production. However, deficiency of peroxisomal FAA2 drastically diminished the yield of {gamma}-decalactone. Thus, this study uncovers the metabolic barriers to oleic acid-to-{gamma}-decalactone conversion and identifies Faa2 as an essential element in this biotransformation.

Glycosylated flavonoids are plant-derived compounds of significant interest due to their enhanced solubility, stability, and bioavailability, offering therapeutic potential across pharmaceutical, nutraceutical, and cosmetic sectors. However, their complex biosynthesis in plants hinders scalable production. In this study, we present an innovative microbial platform based on a phosphate-responsive Pliar53 promoter system in non-model chassis Escherichia coli W to enable efficient, regulated heterologous expression of the SbaiC7OGT gene from Scutellaria baicalensis. This platform circumvents common limitations associated with conventional inducible systems that rely on costly or toxic chemical inducers. The engineered E. coli SBG2413 strain demonstrated high titers of naringenin-7-O-glucoside (prunin) and exhibited broad substrate compatibility for the biosynthesis of other flavonoid glycosides. Our findings establish a cost-effective, scalable solution for industrial production of glycosylated flavonoids, with potential applicability to co-culture systems and microbial consortia. Highlights- A phosphate-response gene expression system (Pliar53) enables stable expression of a toxic flavonoid glycosyltransferase. - Translational optimization using the bicistronic BCD2 RBS and high-copy plasmids boosts prunin production from naringenin. - E. coli W SBG2413strain achieves > 9 g/L prunin in fed-batch bioreactor with 70% conversion. - The platform developed allows efficient glycosylation of structurally diverse flavonoids including flavones, flavanones, and isoflavones.

Consumption of plant-based oils, especially palm oil, is increasing at an alarming rate. This boosted demand for palm oil has drastic effects on the ecosystem as its production is not sustainable. C. oleaginosus is an oleaginous yeast with great potential as a source for microbial-based oil production which is a sustainable alternative to palm oil. However, microbial processes are not yet economically feasible to replace palm oil, unto a large extent due to limited lipid accumulation in the microbe, which limits titers and productivity. Therefore, obtaining enhanced lipid accumulation is essential to render this process commercially viable. Herein we deployed a systematic, iterative Design-Build-Test-Learn (DBTL) approach to establish C. oleaginosus as an efficient fatty acid production platform. In the design step, we identified genes and medium supplements that improved lipid content. To this end, we compared its transcriptional landscape in conditions with high and low amounts of lipid production. A metabolic map was reconstructed and integrated with the expression data. Finally, the genome-scale metabolic model of C. oleaginosus was used to explore metabolism under maximal growth and maximal production conditions. The combination of these four analyses led to the selection of four overexpression targets (ATP-citrate lyase (ACL1), acetyl-CoA carboxylase (ACC), threonine synthase (TS), and hydroxymethylglutaryl-CoA synthase (HMGS)) and five media supplements (biotin, thiamine, threonine, serine, and aspartate). We established an electroporation-based co-transformation method to implement selected genetic interventions. These findings were experimentally validated in the build and test steps of the DBTL approach by adding supplements into the medium and overexpressing the identified genes. Characterization of ACL, ACC, and TS at various C/N ratios, and the addition of medium supplements provided up to 56% (w/w) lipid content, and a 2.5-fold increase in total lipid in the glycerol and urea-based defined medium. In the learn step, quadratic models identified the optimum C/N ratios shifted towards around C/N240. These results firmly confirm C. oleaginous as a sustainable alternative to replace palm as an oil source. HighlightsO_LITranscriptional profile and metabolic model analyzed, predicting genetic targets and medium supplements. C_LIO_LIGenetic targets and medium supplements for improved oil production. C_LIO_LIThe genetic toolbox for C. oleaginosus was expanded (co-transformation method, promoters, genes, and terminators). C_LIO_LIExperimental validations showed that biotin, and threonine increased lipid content. C_LIO_LIOverexpression of ACL1, ACC, and TS in C. oleaginosus provided higher oil content. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/585731v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@e3b6org.highwire.dtl.DTLVardef@65d016org.highwire.dtl.DTLVardef@40952eorg.highwire.dtl.DTLVardef@22724_HPS_FORMAT_FIGEXP M_FIG C_FIG

Glucosinolates are amino acid-derived defense compounds characteristic of the Brassicales order. Benzylglucosinolate (BGLS) derived from phenylalanine is associated with health-promoting effects, which has primed a desire to produce BGLS in microorganisms for a stable and rich source. In this study, we engineered the BGLS production in Saccharomyces cerevisiae by either stably integrating the biosynthetic genes into the genome or introducing them from plasmids. A comparison of the two approaches exhibited a significantly higher level of BGLS production (9.3-fold) by expression of the genes from genome than from plasmids. Towards optimization of BGLS production from genes stably integrated into the genome, we enhanced expression of the entry point enzymes CYP79A2 and CYP83B1 resulting in a 2-fold increase in BGLS production, but also a 4.8-fold increase in the biosynthesis of the last intermediate desulfo-benzylglucosinolate (dsBGLS). To alleviate the metabolic bottleneck in the last step converting dsBGLS to BGLS by 3-phosphoadenosine-5-phosphosulfate (PAPS)-dependent sulfotransferase, SOT16, we first obtained an increased BGLS production by 1.7-fold when overexpressing SOT16. Next, we introduced APS kinase APK1 of Arabidopsis thaliana for efficient PAPS regeneration, which improved the level of BGLS production by 1.7-fold. Our work shows an optimized production of BGLS in S. cerevisiae and the effect of different approaches for engineering the biosynthetic pathway (plasmid expression and genome integration) on the production level of BGLS.

Microbial fermentation for chemical production is becoming more broadly adopted as an alternative to petrochemical refining. Fermentation typically relies on sugar as a feedstock, however, one-carbon compounds like methanol are an attractive alternative as they can be derived from organic waste and natural gas. This study focused on engineering methanol assimilation in the yeast Saccharomyces cerevisiae. Three methanol assimilation pathways were engineered and tested: a synthetic xylulose monophosphate (XuMP), a hybrid methanol dehydrogenase-XuMP, and a bacterial ribulose monophosphate (RuMP) pathway, with the latter identified as the most effective at assimilating methanol. Additionally, 13C-methanol tracer analysis uncovered a native capacity for methanol assimilation in S. cerevisiae, which was optimized using Adaptive Laboratory Evolution. Three independent lineages selected in liquid methanol-yeast extract medium evolved premature stop codons in YGR067C, which encodes an uncharacterised protein that has a predicted DNA-binding domain with homology to the ADR1 transcriptional regulator. Adr1p regulates genes involved in ethanol metabolism and peroxisomal proliferation, suggesting YGR067C has a related function. When one of the evolved YGR067C mutations was reverse engineered into the parental CEN.PK113-5D strain, there were up to 5-fold increases in 13C-labelling of intracellular metabolites from 13C-labelled methanol when 0.1 % yeast extract was a co-substrate, and a 44 % increase in final biomass. Transcriptomics and proteomics revealed that the reconstructed YGR067C mutation results in down-regulation of genes in the TCA cycle, glyoxylate cycle, and gluconeogenesis, which would normally be up-regulated during growth on a non-fermentable carbon source. Combining the synthetic RuMP and XuMP pathways with the reconstructed Ygr067cp truncation led to further improvements in growth. These results identify a latent methylotrophic metabolism in S. cerevisiae and pave the way for further development of native and synthetic one-carbon assimilation pathways in this model eukaryote.

Integrated metabolic engineering approaches combining system and synthetic biology tools allow the efficient designing of microbial cell factories to synthesize high-value products. In the present study, in silico design algorithms were used on the latest yeast genome-scale model 8.5.0 to predict potential genomic modifications that could enhance the production of early-step Taxol(R) in previously engineered Saccharomyces cerevisiae cells. The solution set containing genomic modification candidates was narrowed down by employing the COnstraints Based Reconstruction and Analysis (COBRA) methods. 17 genomic modifications consisting of nine gene deletions and eight gene overexpression were screened using wet-lab studies to determine whether these modifications can increase the production yield of taxadiene, the first metabolite in the Taxol(R) through the mevalonate pathway. Depending on the cultivation condition, most of the single genomic modifications resulted in higher taxadiene production. The best-performing strain, named KM32, contained four overexpressed genes, ILV2, TRR1, ADE13 and ECM31, from the branched-chain amino acid biosynthesis, thioredoxin system, de novo purine synthesis, and the pantothenate pathway, respectively. Using KM32, taxadiene production was increased by 50%, reaching 215 mg/L of taxadiene. The engineered strain also produced 43.65 mg/L of taxa-4(20),11-dien-5-ol (T5-ol), and 26.2 mg/L of taxa-4(20),11-dien-5--yl acetate (T5Ac) which are the highest productions of these early-step Taxol(R) metabolites reported until now in S. cerevisiae. The findings of this study highlight that the use of computational and integrated approaches can ensure determining promising modifications that are difficult to estimate intuitively to develop yeast cell factories.

QS-21, a saponin extract from the Chilean tree Quillaja saponaria, is gaining popularity as a potent vaccine adjuvant, but its production is constrained due to its low natural abundance and complex chemical structure of the triterpenoid saponins, which hinder large-scale production through plant extraction or chemical synthesis. Microbial biosynthesis presents a promising alternative, with Saccharomyces cerevisiae emerging as a desirable host for triterpenoid production. A key step toward microbial QS-21 synthesis is the efficient biosynthesis of its aglycone core, the triterpenoid quillaic acid. However, its efficient production remains limited by challenges of cytochrome P450 enzyme (CYP450s) activity, including cofactor availability and electron transfer efficiency. To address this limitation, we applied a multi-faceted metabolic engineering strategy to optimise CYP450 activity, including CYP450 expression and cytochrome P450 reductase (CPR) selection. Additionally, aligning CYP450 expression with the ethanol phase, enhanced the metabolic flux toward quillaic acid synthesis, leading to an 85-fold increase in titre. Together, these strategies led to a quillaic acid titre of 385 {+/-} 14 mg/L in flask fermentation. Fed-batch bioreactor fermentations increased quillaic acid titre to 471 {+/-} 20 mg/L and significantly increased the selectivity for QA from 32.6% to 65.1% of the total triterpenoids produced. These findings demonstrate the effectiveness of enhancing CYP450 activity through targeted strategies and reveal potential bottlenecks in CYP450 expression, providing valuable insights for future optimization of triterpenoid production in yeast.

Ginsenosides are the primary active metabolites of ginseng and have been used in traditional herbal medicine in Asia for more than 4,000 years. Protopanaxadiol (PPD) is the precursor of dammarane-type ginsenosides, exhibiting different pharmacological activities. However, extraction of PPD from plant material is cumbersome because of its low concentration. Precision fermentation using recombinant yeast is a promising alternative strategy to produce PPD. For increasing PPD production, the medium and fermentation conditions were optimized by a Design of Experiment (DoE) approach. PPD production increased by 7.5-fold in the final cultivation condition compared to the reference condition. The PPD titer reached 1.2 g/L during simple 500 mL shake flask cultivations, the highest PPD production in shake flasks to date. The protocol presented facilitates parallel evaluation of recombinant yeast, thereby contributing to the much-needed sustainable synthesis of the versatile molecule class of triterpenoids.

Discovering natural product biosynthetic pathways from medicinal plants is challenging and laborious, largely due to the complexity of the transcriptomics-driven pathway prediction process. Here we developed a novel approach that captures the protein-level connections between enzymes for pathway discovery with improved accuracy. We proved that heterologous protein-protein interaction screening in yeast enabled the efficient discovery of both dynamic plant enzyme complexes and the pathways they organize. This approach discovered complexes and pathways in the monoterpene indole alkaloid metabolism of a medicinal plant, kratom with high success rate. Screening using a strictosidine {beta}-D-glucosidase (MsSGD1) against 19 medium-chain dehydrogenase/reductases (MsMDRs) identified five MsSGD1-MsMDR complexes. Three out of the five interacting MsMDRs were then proven functional, while the remaining 14 non-interacting candidates did not show obvious activities. The work discovered three branched pathways by combining transcriptomics, metabolomics, and heterologous PPI screening and demonstrated a new plant pathway discovery strategy.

Saccharomyces cerevisiae has been recognised as a convenient host for the production of early precursors to the Taxol(R) anticancer drug. Recent studies have highlighted the harmful impact of oxidative stress as a result of the activity of Taxol(R) first cytochrome P450-reductase enzymes (Taxus spp. CYP725A4-POR). Here, we evolved a new oxidative stress-tolerant yeast strain on galactose, which led to a three-fold higher titre of the CYP725A4 enzyme substrate, taxadiene. We comprehensively analysed the performance of the evolved and parent strain in galactose-limited chemostat cultures before and during oxidative stress induction. Integrating the transcriptomics and metabolite profiling data in an enzyme-constrained genome scale model enabled a more accurate prediction of changes that occurred to biological pathways as a response to/consequence of evolution and oxidative stress. The analyses showed a better performance of the evolved strain with improved respiration and reduced overflow metabolites production. The strain was robust to re-introduction of the oxidative stress, potentially due to the cross-protection mechanism, which contributed to likely better heme, flavin and NADPH availability for an optimal expression of CYP725A4 and POR in yeast. The increased level of taxadiene production has potentially occurred due to the antioxidant properties of taxadiene or as a mechanism to overcome the toxicity of geranylgeranyl diphosphate, the precursor to taxadiene synthase. HighlightsO_LIThe antioxidant properties of taxadiene promotes its production in Saccharomyces cerevisiae C_LIO_LIS. cerevisiae ALE on H2O2 and galactose regulates Flavin, iron and NADPH metabolism as well as carbon and protein recycling pathways through cross-protection and anticipation mechanisms C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/543533v1_fig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1758c64org.highwire.dtl.DTLVardef@543681org.highwire.dtl.DTLVardef@c67339org.highwire.dtl.DTLVardef@b6c56e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Graphical abstract of the study. Figure was created with BioRender.com. C_FIG

Combinatorial approaches in metabolic engineering enable the optimization of multigene pathways, thereby improving product titers. However, the optimization of complex metabolic pathways is hindered by their multiple interactions. Testing all possible combinations of suitable genetic parts is often prevented by the large number of possible variants. A valuable alternative to this is to use statistical design of experiments and linear modeling to collect important information for optimization without testing every possible combination. The shikimate pathway is an example of a complex metabolic pathway involved in the production of aromatic compounds, which are prevalent in industry. In this study, we explore the impact of the modulation of the expression levels of all the genes in the shikimate and para-aminobenzoic acid (pABA) biosynthesis pathways for pABA production (a widely used industrial intermediate) in Pseudomonas putida. We used this approach to select 14 representative strains from a total of 512 possible combinations. We obtained a range of product titers from 2 to 186.2 mg/l. This information was used to guide a second round of strain construction to further increase the production to 232.1 mg/l. Using this strategy, we demonstrate that aroB expression, encoding 3-dehydroquinate synthase, is a significant limiting factor in the production of pABA.

In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 {+/-} 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 {+/-} 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/500214v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1662a84org.highwire.dtl.DTLVardef@717f66org.highwire.dtl.DTLVardef@1633b36org.highwire.dtl.DTLVardef@1c46c7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Natural products acting on our central nervous systems are in utmost demand to fight against pain and mental disorders. Cannabinoids (CBDs) are proven neuroactive agents to treat anxiety, depression, chronic pain diseases, seizure, strokes and neurological disorders. The scarcity of the hemp-sourced CBD products and the prohibitive manufacturing cost limit the wide application of CBDs. Yeast metabolic engineering offers the flexibility to meet the ever-increasing market demand. In this work, we took a retrosynthetic approach and sequentially identified the rate-limiting steps to improve the biosynthesis of the CBD precursor olivetolic acid (OLA) in Yarrowia lipolytica. We debottlenecked the critical enzymatic steps to overcome the supply of hexanoyl-CoA, malonyl-CoA, acetyl-CoA, NADPH and ATPs to redirect carbon flux toward OLA. Implementation of these strategies led to an 83-fold increase in OLA titer in shaking flask experiment. This work may serve as a baseline for engineering CBD biosynthesis in oleaginous yeast species.

Microbial oils, produced by oleaginous microorganisms, offer a sustainable alternative to plant-derived oils. Among these microorganisms, Cutaneotrichosporon oleaginosus attracts attention as a promising microbial cell factory for sustainable oil production due to its capacity to accumulate lipids with a similar composition to palm oil. Although C. oleaginosus can reach higher lipid contents than other oleaginous yeasts, suboptimal lipid yields and productivity limit economic feasibility. Enhanced productivity is necessary to have a feasible microbial oil production process via C. oleaginosus. In this study, we followed a combinatorial approach for strain design and bioprocess development to improve the lipid content and lipid yield. Initially, we deployed a full factorial design with genetic factors (ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), threonine synthase (TS)) and carbon-to-nitrogen ratio (C/N) in the medium. The C/N ratio appeared to have the most impact on oil accumulation. Combined with genetic modifications, lipid content and lipid yield increased by 1.6-fold. In a two-stage fermentation approach at a 2L scale, the triple transformant overexpressing ACL, ACC, and TS outperformed the wild-type by achieving a lipid content of 75.4% (w/w) with lipid productivity of 0.40 g L-1 h-1 and around 0.30 g lipids/g glycerol. In all, we established a cultivation strategy and strain that reached almost the theoretical maximum yield, and highest lipid content reported for a medium containing glycerol as a carbon source. These results strengthen the basis of using C. oleaginous as a platform for microbial oil production, thereby facilitating the development of processes substituting palm oil with a sustainable alternative. HighlightsO_LIFull factorial design allowed selection of the best performer transformant. C_LIO_LIOverexpression of ACL1, ACC, and TS in C. oleaginosus provided higher lipid yield. C_LIO_LITwo-stage fermentation enhanced the lipid content of wild-type and transformant. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/624102v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1371a7aorg.highwire.dtl.DTLVardef@ab5ae1org.highwire.dtl.DTLVardef@1db84caorg.highwire.dtl.DTLVardef@eb19e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nitroaromatic functional groups can impart valuable properties to chemicals and to biological macromolecules including polypeptides. Para-nitro-L-phenylalanine (pN-Phe) is a nitroaromatic amino acid with uses including immune stimulation and fluorescence quenching. As the chemical synthesis of pN-Phe does not follow green chemistry principles and impedes provision of pN-Phe to engineered bacterial cells in some contexts, we sought to design a de novo biosynthetic pathway for pN-Phe in Escherichia coli. To generate the nitro chemical functional group, we identified natural diiron monooxygenases with measurable in vitro and in vivo activity on envisioned amine-containing precursors of para-amino-L-phenylalanine (pA-Phe) and para-aminophenylpyruvate. By expressing one of these N-oxygenase genes together with previously characterized genes for the biosynthesis of pA-Phe, we achieved the synthesis of pN-Phe from glucose. Through further optimization of the chassis, plasmid constructs, and media conditions, we were able to improve the selectivity of pN-Phe biosynthesis, resulting in a maximum titer of 819 {micro}M in rich defined media under shake-flask conditions. These results provide a foundation for the biosynthesis of related nitroaromatic chemicals and for downstream biological applications that could utilize pN-Phe as a building block. HighlightsO_LIPara-nitro-L-phenylalanine (pN-Phe) is a valuable small molecule for its applications in genetic code expansion. C_LIO_LIWe establish de novo biosynthesis of pN-Phe from glucose in E. coli, which is also the first example of a de novo pathway design for an unnatural but commonly used non-standard amino acid. C_LIO_LIWe show the first use of an N-oxygenase enzyme in the de novo synthesis of a nitroaromatic product. C_LIO_LIScreening of natural N-oxygenases and strain engineering resulted in final pN-Phe titers of 820 {+/-} 130 {micro}M in shake flask experiments with rich defined media. C_LI

Rhodosporidium toruloides has emerged as a prominent candidate for producing single-cell oil from cost-effective feedstocks. In this study, the capability of R. toruloides to produce punicic acid (PuA), a representative plant unusual fatty acid, was investigated. The introduction of acyl lipid desaturase and conjugase (PgFADX) allowed R. toruloides to accumulate 3.7% of total fatty acids as PuA. Delta-12 acyl lipid desaturase (PgFAD2) and diacylglycerol acyltransferase 2 were shown to benefit PuA production. The strain with PgFADX and PgFAD2 coexpression accumulated 12% of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask condition. Utilizing wood hydrolysate as the feedstock, this strain produced 6.4% PuA with a titer of 310 mg/L. Taken together, the results demonstrated that R. toruloides could serve as an ideal platform for the production of plant-derived high-value conjugated fatty acid using agricultural and forestry waste as feedstock.

Malonyl-CoA is the major precursor for the biosynthesis of diverse industrially valuable products such as fatty acids/alcohols, flavonoids, and polyketides. However, its intracellular availability is limited in most microbial hosts, hampering the biological synthesis of such chemicals. To address this limitation, we present a multi-level optimization workflow using modern metabolic engineer-ing technologies to systematically increase the malonyl-CoA levels in Pseudomonas putida. The workflow involves the identification of gene downregulations, chassis selection, and optimization of the acetyl-CoA carboxylase complex through ribosome binding site engineering. Computa-tional tools and high-throughput screening with a malonyl-CoA biosensor enabled the rapid eval-uation of numerous genetic targets. Combining the most beneficial targets led to a 5.8-fold en-hancement in the production titer of the valuable polyketide phloroglucinol. This study demon-strates the effective integration of computational and genetic technologies for engineering P. putida, opening new avenues for the development of industrially relevant strains and the investi-gation of fundamental biological questions.

(2S)-Naringenin is a key precursor for biosynthesis of various high-value flavonoids and possesses a variety of nutritional and pharmaceutical properties on human health. Systematic optimization approaches have been employed to improve (2S)-naringenin production in different microbial hosts. However, very few studies have focused on the spatiotemporal distribution of (2S)-naringenin and related pathway intermediate p-coumaric acid, which is an important factor for efficient production. Here, we show that fine-turning of p-coumaric acid synthesis enables alleviated cell burden and improved (2S)-naringenin production in yeast. First, we systematically optimized the (2S)-naringenin biosynthetic pathway by alleviating the bottleneck downstream of p-coumaric acid and increasing malonyl-CoA supply, which improved (2S)-naringenin production but significant amounts of p-coumaric acid still accumulated outside the cell. We further established a dual dynamic control system through combing a malonyl-CoA biosensor regulator and an RNAi strategy, to autonomously control the synthesis of p-coumaric acid and downregulate a pathway competing for malonyl-CoA. The optimized strains remarkably decreased extracellular accumulation of p-coumaric acid and simultaneously improved (2S)- naringenin production. Finally, production of 933 mg/L of (2S)-naringenin could be achieved by using minimal medium with negligible accumulation of p-coumaric acid. Our work highlights the importance of systematic control of pathway intermediates for efficient microbial production of plant natural products.