Heterologous Biosynthesis Research Articles

Synthetic-biology-based discovery of a fungal macrolide from Macrophomina phaseolina.

A synthetic biology approach based on genome mining and heterologous biosynthesis is a powerful tool for discovering novel natural products from a tremendous gene resource. We carried out fungal genome mining guided by a polyketide synthase gene using a public database and found a putative macrolide biosynthetic gene cluster with a highly reducing polyketide synthase gene and a thioesterase gene in Macrophomina phaseolina. Reconstitution of the cluster in Aspergillus oryzae, a model heterologous host for fungal natural product biosynthesis, produced a new 12-membered macrolide, phaseolide A. The absolute stereochemistry was elucidated by vibrational circular dichroism spectroscopy and the crystalline sponge method.

Heterologous Biosynthesis of Type II Polyketide Products Using E. coli

The heterologous biosynthesis of complex natural products has enabled access to polyketide, nonribosomal peptide, isoprenoid, and other compounds with wide-spanning societal value. Though several surrogate host systems exist, Escherichia coli is often a preferred choice due to its rapid growth kinetics and extensive molecular biology protocols. However, a persistent challenge to the utilization of E. coli has been the successful in vivo reconstitution of type II polyketide synthase (PKS) systems. In particular, gene expression of the ketosynthase (KS) components of the minimal PKS has consistently yielded insoluble protein products. In the following report, two type II PKS systems were functionally reconstituted in E. coli. The approach to do so relied upon the utilization of the native transcriptional coupling between the dimeric KS subunits, leading to soluble recombinant protein products and successful polyketide biosynthesis. Resulting strains produced 10 mg/L TW95c and 25 mg/L dehydrorabelomycin. Hence, the strategy offers a new option in the biosynthetic engineering efforts for the heterologous production of type II polyketide products using E. coli.

Insights into Heterologous Biosynthesis of Arteannuin B and Artemisinin in Physcomitrella patens.

Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADS-CYP71AV1-ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase.

Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA), an antitumor triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, is considered as a key precursor for biosynthesizing other ganoderic acids (GAs) with superior antitumor activities. Our previous study identified CYP5150L8 from G. lucidum as a lanosterol oxidase, and achieved heterologous biosynthesis of GA-HLDOA in Saccharomyces cerevisiae. However, low production of GA-HLDOA in either G. lucidum or heterologous host hindered its further investigation and application. In this study, we constructed a dual tunable system for balancing the expression of CYP5150L8 and a Ganoderma P450 reductase iGLCPR, and performed a comprehensive optimization of CYP5150L8 expression, iGLCPR expression, and glycerol usage. Then, we investigated the fermentation behavior of the best strain in optimized condition in flask and achieved 154.45 mg/L GA-HLDOA production, which was 10.7-fold higher compared with previous report. This study may facilitate the wide-spread application of GA-HLDOA and the discovery of unknown cytochrome P450s in downstream GAs biosynthesis.

Synthetic Multienzyme Complexes, Catalytic Nanomachineries for Cascade Biosynthesis In Vivo.

Multienzyme complexes, or metabolons, are assemblies or clusters of sequential enzymes that naturally exist in metabolic pathways. These nanomachineries catalyze the conversion of metabolites more effectively than the freely floating enzymes by minimizing the diffusion of intermediates in vivo. Bioengineers have devised synthetic versions of multienzyme complexes in cells to synergize heterologous biosynthesis, to improve intracellular metabolic flux, and to achieve higher titer of valuable chemical products. Here, we utilized orthogonal protein reactions (SpyCatcher/SpyTag and SnoopCatcher/SnoopTag pairs) to covalently assemble three key enzymes in the mevalonate biosynthesis pathway and showed 5-fold increase of lycopene and 2-fold increase of astaxanthin production in Escherichia coli. The multienzyme complexes are ellipsoidal nanostructures with hollow interior space and uniform thickness and shapes. Intracellular covalent enzyme assembly has yielded catalytic nanomachineries that drastically enlarged the flux of carotenoid biosynthesis in vivo. These studies also deepened our understanding on the complexity of hierarchical enzyme assembly in vivo.

Progress of Heterologous Biosynthesis of Terpenoids in Engineered Corynebacterium glutamicum

Progress of Heterologous Biosynthesis of Terpenoids in Engineered Corynebacterium glutamicum

Biosynthesis of Chondroitin in Engineered Corynebacterium glutamicum.

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum. CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter Ptac, which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase (ldh) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ΔL-CgCAU in a 5-L fed-batch fermentation with a single distribution Mw of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

Heterologous biosynthesis and characterization of a glycocin from a thermophilic bacterium

The genome of the thermophilic bacterium, Aeribacillus pallidus 8, encodes the bacteriocin pallidocin. It belongs to the small class of glycocins and is posttranslationally modified, containing an S-linked glucose on a specific Cys residue. In this study, the pallidocin biosynthetic machinery is cloned and expressed in Escherichia coli to achieve its full biosynthesis and modification. It targets other thermophilic bacteria with potent activity, demonstrated by a low minimum inhibitory concentration (MIC) value. Moreover, the characterized biosynthetic machinery is employed to produce two other glycopeptides Hyp1 and Hyp2. Pallidocin and Hyp1 exhibit antibacterial activity against closely related thermophilic bacteria and some Bacillus sp. strains. Thus, heterologous expression of a glycocin biosynthetic gene cluster including an S-glycosyltransferase provides a good tool for production of hypothetical glycocins encoded by various bacterial genomes and allows rapid in vivo screening.

Potential Natural Food Preservatives and Their Sustainable Production in Yeast: Terpenoids and Polyphenols.

Terpenoids and polyphenols are high-valued plant secondary metabolites. Their high antimicrobial activities demonstrate their huge potential as natural preservatives in the food industry. With the rapid development of metabolic engineering, it has become possible to realize large-scale production of non-native terpenoids and polyphenols by using the generally recognized as safe (GRAS) strain, Saccharomyces cerevisiae, as a cell factory. This review will summarize the major terpenoid and polyphenol compounds with high antimicrobial properties, describe their native metabolic pathways as well as antimicrobial mechanisms, and highlight current progress on their heterologous biosynthesis in S. cerevisiae. Current challenges and perspectives for the sustainable production of terpenoid and polyphenol as natural food preservatives via S. cerevisiae will also be discussed.

Balancing the non-linear rosmarinic acid biosynthetic pathway by modular co-culture engineering

Pathway balancing is a critical and common challenge for microbial biosynthesis using metabolic engineering approaches. Non-linear biosynthetic pathways, such as diverging and converging pathways, are particularly difficult for bioproduction optimization, because they require delicate balancing between all interconnected constituent pathway modules. The emergence of modular co-culture engineering offers a new perspective for biosynthetic pathways modularization and balancing, as the biosynthetic capabilities of individual pathway modules can be coordinated by flexible adjustment of the subpopulation ratio of the co-culture strains carrying the designated modules. This study developed microbial co-cultures composed of multiple metabolically engineered E. coli strains for heterologous biosynthesis of complex natural product rosmarinic acid (RA) whose biosynthesis involves a complex diverging-converging pathway. Our results showed that, compared with the conventional mono-culture strategy, the engineered two-strain co-cultures significantly improved the RA production. Further pathway modularization and balancing in the context of three-strain co-cultures resulted in additional production improvement. Moreover, metabolically engineered co-culture strains utilizing different carbon substrates were recruited to improve the three-strain co-culture stability. The optimized co-culture based on these efforts produced 172 mg/L RA, exhibiting 38-fold biosynthesis improvement over the parent strain used in mono-culture biosynthesis. The findings of this work demonstrate the strong potentials of modular co-culture engineering for overcoming the challenges of complex natural product biosynthesis involving non-linear pathways.

Building Microbial Hosts for Heterologous Production of N-Methylpyrrolinium.

N-Methylpyrrolinium-derived alkaloids like tropane alkaloids, nicotine, and calystegines are valuable plant source specialized metabolites bearing pharmaceutical or biological activity. Microbial synthesis of the critical common intermediate N-methylpyrrolinium would allow for sustainable production of N-methylpyrrolinium-derived alkaloids. Here, we achieve the production of N-methylpyrrolinium both in Escherichia coli and in Saccharomyces cerevisiae by employing the biosynthetic genes derived from three different plants. Specifically, the diamine oxidases (DAOs) from Anisodus acutangulus were first characterized. Then, we produced N-methylpyrrolinium in vitro from l-ornithine via a combination of the three cascade enzymes, ornithine decarboxylase from Erythroxylum coca, putrescine N-methyltransferase from Anisodus tanguticus, and DAOs from A. acutangulus. Construction of the plant biosynthetic pathway in E. coli and S. cerevisiae resulted in de novo bioproduction of N-methylpyrrolinium with titers of 3.02 and 2.07 mg/L, respectively. Metabolic engineering of the yeast strain to produce N-methylpyrrolinium via decreasing the flux to the product catabolism pathway and improving the cofactor supply resulted in a final titer of 17.82 mg/L. This study not only presents the first microbial synthesis of N-methylpyrrolinium but also lays the foundation for heterologous biosynthesis of N-methylpyrrolinium-derived alkaloids. More importantly, the strains constructed herein can serve as important alternative tools for identifying undiscovered pathway enzymes with a synthetic biology strategy.

Heterologous biosynthesis of a fungal macrocyclic polylactone requires only two iterative polyketide synthases.

Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M. theobromae identified an R-PKS gene, men1, and an NR-PKS gene, men2, which both exhibited highest expression levels during the menisporopsin A production phase. These were cloned into separate vectors for heterologous expression in Aspergillus oryzae NSAR1. Unexpectedly, coexpression of the two PKSs alone was sufficient to catalyze the formation of the macrocyclic polylactone, ascotrichalactone A, a structural derivative of menisporopsin A. The unanticipated esterification and cyclolactonization activities could reside in the unusual thioesterase domain of the NR-PKS, which is similar to that of the NRPS catalyzing elongation and cyclization of trilactone in enterobactin biosynthesis and that of modular PKSs catalyzing macrodiolide formation in elaiophylin and conglobatin biosyntheses.

De novo biosynthesis of myricetin, kaempferol and quercetin in Streptomyces albus and Streptomyces coelicolor.

Flavonols are a flavonoid subfamily widely distributed in plants, including several ones of great importance in human and animal diet (apple, tomato, broccoli, onion, beans, tea). These polyphenolic nutraceuticals exert potent antimicrobial (membrane potential disruptors), antioxidant (free-radical scavengers), pharmacokinetic (CYP450 modulators), anti-inflammatory (lipoxygenase inhibitors), antiangiogenic (VEGF inhibitors) and antitumor (cyclin inhibitors) activities. Biotechnological production of these nutraceuticals, for example via heterologous biosynthesis in industrial actinomycetes, is favored since in plants these polyphenols appear as inactive glycosylated derivatives, in low concentrations or as part of complex mixtures with other polyphenolic compounds. In this work, we describe the de novo biosynthesis of three important flavonols, myricetin, kaempferol and quercetin, in the industrially relevant actinomycetes Streptomyces coelicolor and S. albus. De novo biosynthesis of kaempferol, myricetin and quercetin in actinomycetes has not been described before.

Advances in heterologous biosynthesis of plant and fungal natural products by modular co-culture engineering

Heterologous biosynthesis has been long pursued as a viable approach for high efficiency production of natural products with various industrial values. Conventional methods for heterologous biosynthesis use the mono-culture of an engineered microbe for accommodating the whole target biosynthetic pathway to produce the desired product. The emergence of modular co-culture engineering, which divides the pathway between multiple co-culture strains, presents a new perspective to conduct heterologous biosynthesis and improve the bioproduction performance of natural products. This review highlights recent advances in utilizing the modular co-culture engineering approaches to address the challenges of plant and fungal natural product biosynthesis. Potential directions for future research in this promising field are also discussed.

Glycerol improves heterologous biosynthesis of betulinic acid in engineered Yarrowia lipolytica

Glycerol is the main byproduct of the biodiesel production process and can be converted into valuable products by Yarrowia lipolytica. Betulinic acid is a pentacyclic triterpene with a variety of pharmacological properties. Herein we report heterologous biosynthesis of betulinic acid in Y. lipolytica by co-expressing lupeol synthase opAtLUP1, NADPH-cytochrome P450 monooxygenase opCYP716A12, and NADPH-cytochrome P450 reductase opAtCPR1. Initially, the engineered Y. lipolytica produced 0.32 mg/L betulinic acid. This titer was increased to 9.41 mg/L following P450 enzyme fusion and overexpression of key genes from the upstream mevalonate (MVA) pathway. Substitution of glycerol for glucose further enhanced betulinic acid production to 16.98 mg/L, 1.8-fold higher than the titer obtained with glucose. 26.53 mg/L betulinic acid was obtained with 40 g/L glycerol as the sole carbon source, which was comparable to the titer obtained from Saccharomyces cerevisiae with 50 g/L glucose in shake flask cultivation. Glycerol improved betulinic acid titer by increasing the expression of key genes in the MVA pathway, and by increasing the supply of acetyl-CoA. Our study highlights the possible applications of glycerol as carbon source in the production of valuable terpenoids by engineered Y. lipolytica.

Engineering Heterologous Production of Salicylate Glucoside and Glycosylated Variants.

Salicylate 2-O-β-D-glucoside (SAG) is a plant-derived natural product with potential utility as both an anti-inflammatory and as a plant protectant compound. Heterologous biosynthesis of SAG has been established in Escherichia coli through metabolic engineering of the shikimate pathways and introduction of a heterologous biosynthetic step to allow a more directed route to the salicylate precursor. The final SAG compound resulted from the separate introduction of an Arabidopsis thaliana glucosyltransferase enzyme. In this study, a range of heterologous engineering parameters were varied (including biosynthetic pathway construction, expression plasmid, and E. coli strain) for the improvement of SAG specific production in conjunction with a system demonstrating improved plasmid stability. In addition, the glucoside moiety of SAG was systematically varied through the introduction of the heterologous oliose and olivose deoxysugar pathways. Production of analogs was observed for each newly constructed pathway, demonstrating biosynthetic diversification potential; however, production titers were reduced relative to the original SAG compound.

Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus

BackgroundThe yeast Kluyveromyces marxianus is an emerging cell factory for heterologous protein biosynthesis and its use holds tremendous advantages for multiple applications. However, which genes influence the productivity of desired proteins in K. marxianus has so far been investigated by very few studies.ResultsIn this study, we constructed a K. marxianus recombinant (FIM1/Est1E), which expressed the heterologous ruminal feruloyl esterase Est1E as reporter. UV-60Co-γ irradiation mutagenesis was performed on this recombinant, and one mutant (be termed as T1) was screened and reported, in which the productivity of heterologous Est1E was increased by at least tenfold compared to the parental FIM1/Est1E recombinant. Transcriptional perturbance was profiled and presented that the intracellular vesicle trafficking was enhanced while autophagy be weakened in the T1 mutant. Moreover, whole-genome sequencing combined with CRISPR/Cas9 mediated gene-editing identified a novel functional protein Mtc6p, which was prematurely terminated at Tyr251 by deletion of a single cytosine at 755 loci of its ORF in the T1 mutant. We found that deleting C755 of MTC6 in FIM1 led to 4.86-fold increase in the production of Est1E compared to FIM1, while the autophagy level decreased by 47%; on the contrary, when reinstating C755 of MTC6 in the T1 mutant, the production of Est1E decreased by 66% compared to T1, while the autophagy level increased by 124%. Additionally, in the recombinant with attenuated autophagy (i.e., FIM1 mtc6C755Δ and T1) or interdicted autophagy (i.e., FIM1 atg1Δ and T1 atg1Δ), the productivity of three other heterologous proteins was also increased, specifically the heterologous mannase Man330, the β-1,4-endoxylanase XynCDBFV or the conventional EGFP.ConclusionsOur results demonstrated that Mtc6p was involved in regulating autophagy; attenuating or interdicting autophagy would dramatically improve the yields of desired proteins in K. marxianus, and this modulation could be achieved by focusing on the premature mutation of Mtc6p target.

Functional characterization of squalene epoxidase genes in the medicinal plant Tripterygium wilfordii

Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1–4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1–4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii.

Heterologous Biosynthesis of the Fungal Sesquiterpene Trichodermol in Saccharomyces cerevisiae

Trichodermol, a fungal sesquiterpene derived from the farnesyl diphosphate pathway, is the biosynthetic precursor for trichodermin, a member of the trichothecene class of fungal toxins produced mainly by the genera of Trichoderma and Fusarium. Trichodermin is a promising candidate for the development of fungicides and antitumor agents due to its significant antifungal and cytotoxic effects. It can also serve as a scaffold to generate new congeners for structure-activity relationship (SAR) study. We reconstructed the biosynthetic pathway of trichodermol in Saccharomyces cerevisiae BY4741, and investigated the effect of produced trichodermol on the host by de novo RNA sequencing (RNA-Seq) and quantitative Real-time PCR analyses. Co-expression of pESC::FgTRI5 using plasmid pLLeu-tHMGR-UPC2.1 led to trichodiene production of 683 μg L-1, while integration of only the codon-optimized FgTRI5 into the chromosome of yeast improved the production to 6,535 μg L-1. Subsequent expression of the codon-optimized cytochrome P450 monooxygenase encoding genes, TaTRI4 and TaTRI11, resulted in trichodermol, with an estimated titer of 252 μg L-1 at shake flask level. RNA-Seq and qPCR analyses revealed that the produced trichodermol downregulated the expression of the genes involved in ergosterol biosynthesis, but significantly upregulated the expression of PDR5 related to membrane transport pathway in S. cerevisiae. Collectively, we achieved the first heterologous biosynthesis of trichodermol by reconstructing its biosynthetic pathway in yeast, and the reconstructed pathway will serve as a platform to generate trichodermin analogs as potential candidates for agrochemicals and anticancer agents through further optimizations.

Applications of Yeast Synthetic Biology Geared towards the Production of Biopharmaceuticals.

Engineered yeast are an important production platform for the biosynthesis of high-value compounds with medical applications. Recent years have witnessed several new developments in this area, largely spurred by advances in the field of synthetic biology and the elucidation of natural metabolic pathways. This minireview presents an overview of synthetic biology applications for the heterologous biosynthesis of biopharmaceuticals in yeast and demonstrates the power and potential of yeast cell factories by highlighting several recent examples. In addition, an outline of emerging trends in this rapidly-developing area is discussed, hinting upon the potential state-of-the-art in the years ahead.