Systems Metabolic Engineering Research Articles

Metabolic engineering of Escherichia coli for the efficient production of 5-hydroxyvaleric acid

There is increasing industrial demand for five-carbon platform chemicals, particularly 5-hydroxyvaleric acid (5-HV), a widely used building block for the synthesis of polyesters and polyurethanes. Here, we developed an efficient 5-HV microbial producer by systems metabolic engineering of Escherichia coli W3110. To derive 5-HV, Pseudomonas putidal-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA), E. coli 4-aminobutyrate aminotransferase (gabT), and different aldehyde reductase genes were introduced to E. coli W3110 to convert l-lysine hydrochloride to the target product. Three aldehyde reductases, including YajO from P. putida and YahK and YqhD from E. coli, were examined, revealing that the overexpression of aldehyde reductases was critical for 5-HV production and YajO was first reported for efficient production of 5-HV. The E. coli 5-aminovalerate exporter encoded by gabP and E. coli lysine-specific permease encoded by lysP were expressed to further enhance 5-HV production. In addition, a hok/sok toxin/antitoxin system based on the E. coli XL1-Blue genome was inserted to the dual-vector system to decrease plasmid loss. Finally, 5.59 g/L of 5-HV with a yield of 0.96 mol/mol (5-HV/l-lysine hydrochloride) by shake flask cultivation of the engineered E. coli W3110 and 35.6 g/L of 5-HV with a yield of 0.92 mol/mol (5-HV/l-lysine hydrochloride) by fed fermentation were achieved respectively. The developed metabolic engineering strategies provide a basis for the efficient and sustainable bio-based production of 5-HV.

Efficient Synthesis of Limonene in Saccharomyces cerevisiae Using Combinatorial Metabolic Engineering Strategies

Limonene is a volatile monoterpene compound that is widely used in food additives, pharmaceutical products, fragrances, and toiletries. We herein attempted to perform efficient biosynthesis of limonene in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, we conducted de novo synthesis of limonene in S. cerevisiae and achieved a titer of 46.96 mg/L. Next, by dynamic inhibition of the competitive bypass of key metabolic branches regulated by ERG20 and optimization of the copy number of tLimS, a greater proportion of the metabolic flow was directed toward limonene synthesis, achieving a titer of 640.87 mg/L. Subsequently, we enhanced the acetyl-CoA and NADPH supply, which increased the limonene titer to 1097.43 mg/L. Then, we reconstructed the limonene synthesis pathway in the mitochondria. Dual regulation of cytoplasmic and mitochondrial metabolism further increased the limonene titer to 1586 mg/L. After optimization of the process of fed-batch fermentation, the limonene titer reached 2.63 g/L, the highest ever reported in S. cerevisiae.

System metabolic engineering of Escherichia coli W for the production of 2-ketoisovalerate using unconventional feedstock.

Replacing traditional substrates in industrial bioprocesses to advance the sustainable production of chemicals is an urgent need in the context of the circular economy. However, since the limited degradability of non-conventional carbon sources often returns lower yields, effective exploitation of such substrates requires a multi-layer optimization which includes not only the provision of a suitable feedstock but the use of highly robust and metabolically versatile microbial biocatalysts. We tackled this challenge by means of systems metabolic engineering and validated Escherichia coli W as a promising cell factory for the production of the key building block chemical 2-ketoisovalerate (2-KIV) using whey as carbon source, a widely available and low-cost agro-industrial waste. First, we assessed the growth performance of Escherichia coli W on mono and disaccharides and demonstrated that using whey as carbon source enhances it significantly. Second, we searched the available literature and used metabolic modeling approaches to scrutinize the metabolic space of E. coli and explore its potential for overproduction of 2-KIV identifying as basic strategies the block of pyruvate depletion and the modulation of NAD/NADP ratio. We then used our model predictions to construct a suitable microbial chassis capable of overproducing 2-KIV with minimal genetic perturbations, i.e., deleting the pyruvate dehydrogenase and malate dehydrogenase. Finally, we used modular cloning to construct a synthetic 2-KIV pathway that was not sensitive to negative feedback, which effectively resulted in a rerouting of pyruvate towards 2-KIV. The resulting strain shows titers of up to 3.22 ± 0.07g/L of 2-KIV and 1.40 ± 0.04g/L of L-valine in 24h using whey in batch cultures. Additionally, we obtained yields of up to 0.81g 2-KIV/g substrate. The optimal microbial chassis we present here has minimal genetic modifications and is free of nutritional autotrophies to deliver high 2-KIV production rates using whey as a non-conventional substrate.

The enhanced production and secretion of high-value cajaninstilbene acid and flavonoid aglycones in Cajanus cajan (Linn.) Millsp. cell suspension cultures elicited by Aspergillus oryzae Y-29

For the first time, Cajanus cajan cell suspension cultures (CCSCs) were elicited by living GRAS (Generally Recognized as Safe) fungi to enhance the production of bioactive phenolic compounds. Under the inoculum size of mycelia (0.5 %), the optimal fungus Aspergillus oryzae Y-29 (AO Y-29) could achieve the enhanced production of cajaninstilbene acid (5.79-fold increase) in 49-day-old CCSCs at 8 h. Also, AO Y-29 could promote the production of flavonoid aglycones including luteolin (2.25-fold increase), genistein (2.04-fold increase), apigenin (1.96-fold increase), isorhamnetin (1.43-fold increase), and biochanin A (6.48-fold increase) in CCSCs at 12 h. Moreover, the levels of genistein and cajaninstilbene acid in culture media after fungal elicitation increased by 3.58- and 18.02-fold over control, respectively. AO Y-29 mycelia was found to trigger the oxidative stress thus activating the expression of pathogenesis-, biosynthesis-, and ABC transport-related genes, which contributed to the accumulation and secretion of flavonoid aglycones and cajaninstilbene acid in CCSCs. Furthermore, ABCB11 was likely to be correlated with the transport of genistein and cajaninstilbene acid, which was important for systematic metabolic engineering to increase their productivity. Overall, the elicitation of CCSCs with AO Y-29 was attractive for obtaining high yields of valuable flavonoid aglycones and cajaninstilbene acid in pharmaceutical/nutraceutical fields.

Systems metabolic engineering upgrades Corynebacterium glutamicum for selective high-level production of the chiral drug precursor and cell-protective extremolyte L-pipecolic acid

The nonproteinogenic cyclic metabolite l-pipecolic acid is a chiral precursor for the synthesis of various commercial drugs and functions as a cell-protective extremolyte and mediator of defense in plants, enabling high-value applications in the pharmaceutical, medical, cosmetic, and agrochemical markets. To date, the production of the compound is unfavorably fossil-based. Here, we upgraded the strain Corynebacterium glutamicum for l-pipecolic acid production using systems metabolic engineering. Heterologous expression of the l-lysine 6-dehydrogenase pathway, apparently the best route to be used in the microbe, yielded a family of strains that enabled successful de novo synthesis from glucose but approached a limit of performance at a yield of 180 mmol mol−1. Detailed analysis of the producers at the transcriptome, proteome, and metabolome levels revealed that the requirements of the introduced route were largely incompatible with the cellular environment, which could not be overcome after several further rounds of metabolic engineering. Based on the gained knowledge, we based the strain design on l-lysine 6-aminotransferase instead, which enabled a substantially higher in vivo flux toward l-pipecolic acid. The tailormade producer C. glutamicum PIA-7 formed l-pipecolic acid up to a yield of 562 mmol mol−1, representing 75% of the theoretical maximum. Ultimately, the advanced mutant PIA-10B achieved a titer of 93 g L−1 in a fed-batch process on glucose, outperforming all previous efforts to synthesize this valuable molecule de novo and even approaching the level of biotransformation from l-lysine. Notably, the use of C. glutamicum allows the safe production of GRAS-designated l-pipecolic acid, providing extra benefit toward addressing the high-value pharmaceutical, medical, and cosmetic markets. In summary, our development sets a milestone toward the commercialization of biobased l-pipecolic acid.

Identification of key genes involved in secondary metabolite biosynthesis in Digitalis purpurea.

The medicinal plant Digitalis purpurea produces cardiac glycosides that are useful in the pharmaceutical industry. These bioactive compounds are in high demand due to ethnobotany's application to therapeutic procedures. Recent studies have investigated the role of integrative analysis of multi-omics data in understanding cellular metabolic status through systems metabolic engineering approach, as well as its application to genetically engineering metabolic pathways. In spite of numerous omics experiments, most molecular mechanisms involved in metabolic pathways biosynthesis in D. purpurea remain unclear. Using R Package Weighted Gene Co-expression Network Analysis, co-expression analysis was performed on the transcriptome and metabolome data. As a result of our study, we identified transcription factors, transcriptional regulators, protein kinases, transporters, non-coding RNAs, and hub genes that are involved in the production of secondary metabolites. Since jasmonates are involved in the biosynthesis of cardiac glycosides, the candidate genes for Scarecrow-Like Protein 14 (SCL14), Delta24-sterol reductase (DWF1), HYDRA1 (HYD1), and Jasmonate-ZIM domain3 (JAZ3) were validated under methyl jasmonate treatment (MeJA, 100 μM). Despite early induction of JAZ3, which affected downstream genes, it was dramatically suppressed after 48 hours. SCL14, which targets DWF1, and HYD1, which induces cholesterol and cardiac glycoside biosynthesis, were both promoted. The correlation between key genes and main metabolites and validation of expression patterns provide a unique insight into the biosynthesis mechanisms of cardiac glycosides in D. purpurea.

Lignin Valorization: Production of High Value-Added Compounds by Engineered Microorganisms

Lignin is the second most abundant polymer in nature, which is also widely generated during biomass fractionation in lignocellulose biorefineries. At present, most of technical lignin is simply burnt for energy supply although it represents the richest natural source of aromatics, and thus it is a promising feedstock for generation of value-added compounds. Lignin is heterogeneous in composition and recalcitrant to degradation, with this substantially hampering its use. Notably, microbes have evolved particular enzymes and specialized metabolic pathways to degrade this polymer and metabolize its various aromatic components. In recent years, novel pathways have been designed allowing to establish engineered microbial cell factories able to efficiently funnel the lignin degradation products into few metabolic intermediates, representing suitable starting points for the synthesis of a variety of valuable molecules. This review focuses on recent success cases (at the laboratory/pilot scale) based on systems metabolic engineering studies aimed at generating value-added and specialty chemicals, with much emphasis on the production of cis,cis-muconic acid, a building block of recognized industrial value for the synthesis of plastic materials. The upgrade of this global waste stream promises a sustainable product portfolio, which will become an industrial reality when economic issues related to process scale up will be tackled.

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.

Improving the production of the micafungin precursor FR901379 in an industrial production strain

BackgroundMicafungin is an echinocandin-type antifungal agent used for the clinical treatment of invasive fungal infections. It is semisynthesized from the sulfonated lipohexapeptide FR901379, a nonribosomal peptide produced by the filamentous fungus Coleophoma empetri. However, the low fermentation efficiency of FR901379 increases the cost of micafungin production and hinders its widespread clinical application.ResultsHere, a highly efficient FR901379-producing strain was constructed via systems metabolic engineering in C. empetri MEFC09. First, the biosynthesis pathway of FR901379 was optimized by overexpressing the rate-limiting enzymes cytochrome P450 McfF and McfH, which successfully eliminated the accumulation of unwanted byproducts and increased the production of FR901379. Then, the functions of putative self-resistance genes encoding β-1,3-glucan synthase were evaluated in vivo. The deletion of CEfks1 affected growth and resulted in more spherical cells. Additionally, the transcriptional activator McfJ for the regulation of FR901379 biosynthesis was identified and applied in metabolic engineering. Overexpressing mcfJ markedly increased the production of FR901379 from 0.3 g/L to 1.3 g/L. Finally, the engineered strain coexpressing mcfJ, mcfF, and mcfH was constructed for additive effects, and the FR901379 titer reached 4.0 g/L under fed-batch conditions in a 5 L bioreactor.ConclusionsThis study represents a significant improvement for the production of FR901379 and provides guidance for the establishment of efficient fungal cell factories for other echinocandins.

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.

High-Level Productivity of Lacto-N-neotetraose in Escherichia coli by Systematic Metabolic Engineering.

Lacto-N-neotetraose (LNnT) is a critical component of human milk oligosaccharides. This study introduces a systems metabolic engineering method to produce LNnT in Escherichia coli. First, 12 target genes contributing to LNnT production were identified using a double-plasmid system. Subsequently, combinatorial optimization of the copy number was performed to tune the target gene expression strength. Next, the CRISPR/Cas9 system was used to block the UDP-Gal and UDP-GlcNAc competitive pathways, and the titer of LNnT reached 1.16 g/L (E27). Moreover, the lactoylglutathione lyase (GloA) was deleted to block the competing metabolite pathway from glycerol to lactate, and the titer of LNnT (1.46 g/L) was 26% higher than that of strain E27. Finally, the LNnT productivity was increased to 0.34 g/L/h in a 3 L bioreactor, which was 36% higher than the recently reported LNnT productivity. This research work opens an innovative framework for the effective production of LNnT.

Cocultivation of pigeon pea hairy root cultures and Aspergillus for the enhanced production of cajaninstilbene acid.

Pigeon pea hairy root cultures (PPHRCs) have been proven to be a promising alternative for the production of health-beneficial phenolic compounds, such as the most important health-promoting compound, i.e., cajaninstilbene acid (CSA). In this study, PPHRCs were cocultured with live Aspergillus fungi for further improving phenolic productivity via biological elicitation. Aspergillus oryzae CGMCC 3.951 (AO 3.951) was found to be the optimal fungus that could achieve the maximum increment of CSA (10.73-fold increase) in 42-day-old PPHRCs under the inoculum size of mycelia 0.50% and cocultivation time 36h. More precisely, the contents of CSA in hairy roots and culture media after fungal elicitation increased by 9.87- and 62.18-fold over control, respectively. Meanwhile, the contents of flavonoid glycosides decreased, while aglycone yields increased upon AO 3.951 elicitation. Moreover, AO 3.951 could trigger the oxidative stress and pathogen defense response thus activating the expression of biosynthesis- and ABC transporter-related genes, which contributed to the intracellular accumulation and extracellular secretion of phenolic compounds (especially CSA) in PPHRCs. And PAL2, 4CL2, STS1, and I3'H were likely to be the potential key enzyme genes regulating the biosynthesis of CSA, and ABCB11X1-1, ABCB11, and ABCG24X2 were closely related to the transmembrane transport of CSA. Overall, the cocultivation approach could make PPHRCs more commercially attractive for the production of high-value phenolic compounds such as CSA and flavonoid aglycones in nutraceutical/medicinal fields. And the elucidation of crucial biosynthesis and transport genes was important for systematic metabolic engineering aimed at increasing CSA productivity. KEY POINTS: • Cocultivation of PPHRCs and live fungi was to enhance CSA production and secretion. • PPHRCs augmented CSA productivity 10.73-fold when cocultured with AO 3.951 mycelia. • Several biosynthesis and transport genes related to CSA production were clarified.

Systematic metabolic engineering of Escherichia coli for the enhanced production of cinnamaldehyde

Cinnamaldehyde (CAD) derived from cinnamon bark has received much attention for its potential as a nematicide and food additive. Previously, we have succeeded in developing an Escherichia coli strain (YHP05) capable of synthesizing cinnamaldehyde; however, the production titer (75 mg/L) was not sufficient for commercialization. Herein, to develop an economical and sustainable production bioprocess, we further engineered the YHP05 strain for non-auxotrophic, antibiotic-free, inducer-free hyperproduction of CAD using systematic metabolic engineering. First, the conversion of trans-cinnamic acid (t-CA) to CAD was improved by the co-expression of carboxylic acid reductase and phosphopantetheinyl transferase (PPTase) genes. Second, to prevent the spontaneous conversion of CAD to cinnamyl alcohol, 10 endogenous reductase and dehydrogenase genes were deleted. Third, all expression cassettes were integrated into the chromosomal DNA using an auto-inducible system for antibiotic- and inducer-free production. Subsequently, to facilitate CAD production, available pools of cofactors (NADPH, CoA, and ATP) were increased, and acetate pathways were deleted. With the final antibiotic-, plasmid-, and inducer-free strain (H-11MPmR), fed-batch cultivations combined with in situ product recovery (ISPR) were performed, and the production titer of CAD as high as 3.8 g/L could be achieved with 49.1 mg/L/h productivity, which is the highest CAD titer ever reported.

Reprogramming the metabolism of oleaginous yeast for sustainably biosynthesizing the anticarcinogen precursor germacrene A

The highest titer of the anticancer precursor sesquiterpene germacrene A was observed in oleaginous yeast using multi-layered systematic metabolic engineering strategies.

Improving methanol assimilation in Yarrowia lipolytica via systematic metabolic engineering combined with compartmentalization

Synthetic methylotrophic Yarrowia lipolytica was constructed to convert methanol into biomass components and succinic acid.

Systematic metabolic engineering of Klebsiella oxytoca for production of 1,3-propanediol from glucose

This study successfully engineered K. oxytoca to produce 1,3-PDO from glucose for the first time.

Biosynthesis of α-bisabolene from low-cost renewable feedstocks by peroxisome engineering and systems metabolic engineering of the yeast Yarrowia lipolytica

Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and insufficient precursor supply.

Systems metabolic engineering upgrades Corynebacterium glutamicum to high-efficiency cis, cis-muconic acid production from lignin-based aromatics

Lignin displays a highly challenging renewable. To date, massive amounts of lignin, generated in lignocellulosic processing facilities, are for the most part merely burned due to lacking value-added alternatives. Aromatic lignin monomers of recognized relevance are in particular vanillin, and to a lesser extent vanillate, because they are accessible at high yield from softwood-lignin using industrially operated alkaline oxidative depolymerization. Here, we metabolically engineered C. glutamicum towards cis, cis-muconate (MA) production from these key aromatics. Starting from the previously created catechol-based producer C. glutamicum MA-2, systems metabolic engineering first discovered an unspecific aromatic aldehyde reductase that formed aromatic alcohols from vanillin, protocatechualdehyde, and p- hydroxybenzaldehyde, and was responsible for the conversion up to 57% of vanillin into vanillyl alcohol. The alcohol was not re-consumed by the microbe later, posing a strong drawback on the producer. The identification and subsequent elimination of the encoding fudC gene completely abolished vanillyl alcohol formation. Second, the initially weak flux through the native vanillin and vanillate metabolism was enhanced up to 2.9-fold by implementing synthetic pathway modules. Third, the most efficient protocatechuate decarboxylase AroY for conversion of the midstream pathway intermediate protocatechuate into catechol was identified out of several variants in native and codon optimized form and expressed together with the respective helper proteins. Fourth, the streamlined modules were all genomically combined which yielded the final strain MA-9. MA-9 produced bio-based MA from vanillin, vanillate, and seven structurally related aromatics at maximum selectivity. In addition, MA production from softwood-based vanillin, obtained through alkaline depolymerization, was demonstrated.

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine

The host-guest incompatibility between a production host and non-native enzymes has posed an arduous challenge for synthetic biology, particularly between mesophile-derived enzymes and a thermophilic chassis. In the present study, we develop a thermophilic enzyme mining strategy comprising an automated co-evolution-based screening pipeline (http://cem.sjtu.edu.cn), computation-based enzyme characterization, and gene synthesis-based function validation. Using glucosamine-6-phosphate acetyltransferase (GNA1) as an example, we successfully mined four novel GNA1s with excellent thermostabilities and catalytic performances. Calculation and analysis based on AlphaFold2-generated structures were also conducted to uncover the mechanism underlying their excellent properties. Finally, our mined GNA1s were used to enable the high-temperature N-acetylglucosamine (GlcNAc) production with high titers of up to 119.3g/L, with the aid of systems metabolic engineering and temperature programming. This study demonstrates the effectiveness of the enzyme mining strategy, highlighting the application prospects of mining new enzymes from massive databases and providing an effective solution for tackling host-guest incompatibility.

Engineering Gluconobacter cerinus CGMCC 1.110 for direct 2-keto-L-gulonic acid production.

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9g/L.