Combinatorial Engineering Strategies Research Articles

De novo biosynthesis of β-Arbutin in Komagataella phaffii based on metabolic engineering strategies

Backgroundβ-Arbutin, found in the leaves of bearberry, stands out as one of the globally acknowledged eco-friendly whitening additives in recent years. However, the natural abundance of β-Arbutin is low, and the cost-effectiveness of using chemical synthesis or plant extraction methods is low, which cannot meet the requirements. While modifying the β-Arbutin synthesis pathway of existing strains is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.ResultsIn this study, we established a biosynthetic pathway in Komagataella phaffii for β-Arbutin production with a titer of 1.58 g/L. Through diverse metabolic strategies, including fusion protein construction, enhancing shikimate pathway flux, and augmenting precursor supplies (PEP, E4P, and UDPG), we significantly increased β-Arbutin titer to 4.32 g/L. Further optimization of methanol concentration in shake flasks led to a titer of 6.32 g/L titer after 120 h of fermentation, representing a fourfold increase over the initial titer. In fed-batch fermentation, strain UA3-10 set a record with the highest production to date, reaching 128.6 g/L in a 5 L fermenter.ConclusionsThis is the highest yield in the fermentation tank level of using microbial cell factories for de novo synthesis of β-Arbutin. Applying combinatorial engineering strategies has significantly improved the β-Arbutin yield in K. phaffii and is a promising approach for synthesizing functional products using a microbial cell factory. This study not only advances low-cost fermentation-based production of β-Arbutin but also establishes K. phaffii as a promising chassis cell for synthesizing other aromatic amino acid metabolites.Graphical abstract

Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies

BackgroundLimonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.ResultsIn this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce d- and l-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of l-limonene and 24.8 mg/L of d-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of d-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased d-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, d-limonene production titer reached 69.3 mg/L.ConclusionsIn this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.

Metabolic engineering of Saccharomyces cerevisiae for chelerythrine biosynthesis

BackgroundChelerythrine is an important alkaloid used in agriculture and medicine. However, its structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from plants. Here, we reconstructed and optimized the complete biosynthesis pathway for chelerythrine from (S)-reticuline in Saccharomyces cerevisiae using genetic reprogramming.ResultsThe first-generation strain Z4 capable of producing chelerythrine was obtained via heterologous expression of seven plant-derived enzymes (McoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, and PsCPR) in S. cerevisiae W303-1 A. When this strain was cultured in the synthetic complete (SC) medium supplemented with 100 µM of (S)-reticuline for 10 days, it produced up to 0.34 µg/L chelerythrine. Furthermore, efficient metabolic engineering was performed by integrating multiple-copy rate-limiting genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, INO2, and AtATR1), tailoring the heme and NADPH engineering, and engineering product trafficking by heterologous expression of MtABCG10 to enhance the metabolic flux of chelerythrine biosynthesis, leading to a nearly 900-fold increase in chelerythrine production. Combined with the cultivation process, chelerythrine was obtained at a titer of 12.61 mg per liter in a 0.5 L bioreactor, which is over 37,000-fold higher than that of the first-generation recombinant strain.ConclusionsThis is the first heterologous reconstruction of the plant-derived pathway to produce chelerythrine in a yeast cell factory. Applying a combinatorial engineering strategy has significantly improved the chelerythrine yield in yeast and is a promising approach for synthesizing functional products using a microbial cell factory. This achievement underscores the potential of metabolic engineering and synthetic biology in revolutionizing natural product biosynthesis.

Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Enhancing the Production of Xenocoumacin 1 in Xenorhabdus nematophila CB6 by a Combinatorial Engineering Strategy.

Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against Phytophthora capsici. However, the commercial development of Xcn1 is hindered by the low yield, which results in high application costs. In this study, multiple metabolic strategies, including blocking the degradation pathway, promoter engineering, and deletion of competing biosynthetic gene clusters, were employed to improve the production of Xcn1, which was increased from 0.07 to 0.91 g/L. The formation of Xcn1 reached 1.94 g/L in the TB medium with the final strain T3 in a shake flask and further reached 3.52 g/L in a 5 L bioreactor, which is the highest yield ever reported. The engineered strain provides a valuable platform for production of Xcn1, and the possible commercial development of the biofungicide. We anticipate that the metabolic engineering strategies utilized in this study and the constructed constitutive promoter library can be widely applied to other bacteria of the genera Xenorhabdus and Photorhabdus.

Identification of Fcl-29 as an Effective Antifungal Natural Product against Fusarium graminearum and Combinatorial Engineering Strategy for Improving Its Yield.

Fusarium head blight (FHB), caused by Fusarium graminearum, whose occurrence and prevalence causes 10-70% wheat production loss, is one of the most destructive diseases influencing the production of wheat globally. To identify the potential natural products (NPs) against F. graminearum, we screened 59 Xenorhabdus strains and discovered that the cell-free supernatant (CFS) of X. budapestensis 14 (XBD14) displays the highest bioactivity. Multiple genetic methods coupled with HRMS/MS analysis determined the major antifungal NP to be Fcl-29, a fabclavine derivative. Fcl-29 was found to effectively control FHB of wheat in the field test and demonstrated broad-spectrum antifungal activity against important pathogenic fungi. The production of Fcl-29 was dramatically improved by 33.82-fold with the combinatorial strategy of genetic engineering (1.66-fold) and fermentation engineering (20.39-fold). The exploration of a new biofungicide in global plant protection is now possible.

Construction of cell factory through combinatorial metabolic engineering for efficient production of itaconic acid

BackgroundItaconic acid, an unsaturated C5 dicarbonic acid, has significant market demand and prospects. It has numerous biological functions, such as anti-cancer, anti-inflammatory, and anti-oxidative in medicine, and is an essential renewable platform chemical in industry. However, the development of industrial itaconic acid production by Aspergillus terreus, the current standard production strain, is hampered by the unavoidable drawbacks of that species. Developing a highly efficient cell factory is essential for the sustainable and green production of itaconic acid.ResultsThis study employed combinatorial engineering strategies to construct Escherichia coli cells to produce itaconic acid efficiently. Two essential genes (cis-aconitate decarboxylase (CAD) encoding gene cadA and aconitase (ACO) encoding gene acn) employed various genetic constructs and plasmid combinations to create 12 recombination E. coli strains to be screened. Among them, E. coli BL-CAC exhibited the highest titer with citrate as substrate, and the induction and reaction conditions were further systematically optimized. Subsequently, employing enzyme evolution to optimize rate-limiting enzyme CAD and synthesizing protein scaffolds to co-localize ACO and CAD were used to improve itaconic acid biosynthesis efficiency. Under the optimized reaction conditions combined with the feeding control strategy, itaconic acid titer reached 398.07 mM (51.79 g/L) of engineered E. coli BL-CAR470E-DS/A-CS cells as a catalyst with the highest specific production of 9.42 g/g(DCW) among heterologous hosts at 48 h.ConclusionsThe excellent catalytic performance per unit biomass shows the potential for high-efficiency production of itaconic acid and effective reduction of catalytic cell consumption. This study indicates that it is necessary to continuously explore engineering strategies to develop high-performance cell factories to break through the existing bottleneck and achieve the economical commercial production of itaconic acid.

Enhancing heterologous expression of a key enzyme for the biosynthesis of 2'-fucosyllactose.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide (HMO) in human milk and has important physiological functions. The market demand of 2'-FL is continuing to grow, but high production cost has limited its availability. To solve the dilemma, biosynthesis of 2'-FL has been proposed and is considered the most promising pathway for massive production. α-1,2-Fucosyltransferase is one of the key elements involved in its biosynthesis, but the limited intracellular accumulation and unstable properties of α-1,2-fucosyltransferases when expressed in host strains have become a major hurdle for the effective biosynthesis of 2'-FL. A combinatorial engineering strategy of synergic modification of ribosome binding site, fusion peptide and enzyme gene was leveraged to enhance the soluble expression of α-1,2-fucosyltransferases and promote enzyme activity. The preferable combination was to employ an optimized ribosome binding site region to drive 3 × FLAG as a fusion partner along with the α-1,2-fucosyltransferase for expression in Escherichia coli (DE3) PlySs, and protein yield and enzyme activity were remarkably improved by 11.51-fold and 13.72-fold, respectively. After finely tuning the synergy among different elements, the abundant protein yield and high enzyme activity confirmed that the drawbacks of heterologous expression in α-1,2-fucosyltransferase had been properly addressed. A suitable external environment further drives the efficient synthesis of α-1,2-fucosyltransferases. To our knowledge, this is the first report of a systematic and effective modification of α-1,2-fucosyltransferase expression, which could potentially serve as a guideline for industrial application. © 2022 Society of Chemical Industry.

Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid.

As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported for the biosynthesis of caffeic acid via episomal plasmid-mediated expression of pathway genes. However, the production was far from satisfactory and even relied on the addition of precursor. In this study, we first established a controllable and stable caffeic acid pathway by employing a modified GAL regulatory system to control the genome-integrated pathway genes in S. cerevisiae and realized biosynthesis of 222.7 mg/L caffeic acid. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways, and overexpressing rate-limiting enzymes led to about 2.6-fold improvement in the caffeic acid production, reaching up to 569.0 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid synthesized by engineered yeast. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites. KEY POINTS: Genomic integration of ORgTAL, OHpaB, and HpaC for caffeic acid production in yeast. Feedback inhibition elimination and Aro10 deletion improved caffeic acid production. The highest ever reported titer (569.0 mg/L) of caffeic acid synthesized by yeast.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

Genome-based rational engineering of Actinoplanes deccanensis for improving fidaxomicin production and genetic stability

Microbial fermentation is currently still the major way to produce structural complicated clinical drugs. Yet, the low productivity and genetic instability of producing strains remain the bottlenecks in microbial pharmaceutical industry. Fidaxomicin is a microbial drug against the Clostridium difficile infection. Here, a genome-based combinatorial engineering strategy was established to improve both fidaxomicin production and the genetic stability of Actinoplanes deccanensis YP-1. Guided by genomic analysis, several genetic instability-associated elements were cumulatively deleted, generating a more genetically stable mutant. Further rational engineering approaches including elimination of a pigment pathway, duplication of the fidaxomicin gene cluster, overexpression of a positive regulator and optimization of the fermentation medium, led to an overall 27-folds improvement in fidaxomicin production. Taken together, the genome-based rational combinatorial engineering strategy was efficient to enhance the fidaxomicin production and ameliorate the genetic stability of YP-1, it can also be widely used in other industrial actinomycetes for strain improvement.

Modulating Pathway Performance by Perturbing Local Genetic Context.

Combinatorial engineering is a preferred strategy for attaining optimal pathway performance. Previous endeavors have been concentrated on regulatory elements (e.g., promoters, terminators, and ribosomal binding sites) and/or open reading frames. Accumulating evidence indicates that noncoding DNA sequences flanking a transcriptional unit on the genome strongly impact gene expression. Here, we sought to mimic the effect imposed on expression cassettes by the genome. We created variants of the model yeast Saccharomyces cerevisiae with significantly improved fluorescence or cellobiose consumption rate by randomizing the sequences adjacent to the GFP expression cassette or the cellobiose-utilization pathway, respectively. Interestingly, nucleotide specificity was observed at certain positions and showed to be essential for achieving optimal cellobiose assimilation. Further characterization suggested that the modulation effects of the short sequences flanking the expression cassettes could be potentially mediated by remodeling DNA packaging and/or recruiting transcription factors. Collectively, these results indicate that the often-overlooked contiguous DNA sequences can be exploited to rapidly achieve balanced pathway expression, and the corresponding approach could be easily stacked with other combinatorial engineering strategies.

High-level production of linalool by engineered Saccharomyces cerevisiae harboring dual mevalonate pathways in mitochondria and cytoplasm

Linalool, a valuable monoterpene alcohol, is widely used in cosmetics and flavoring ingredients. However, its scalable production by microbial fermentation is not yet achieved. In this work, considerable increase in linalool production was obtained in Saccharomyces cerevisiae by dual metabolic engineering of the mevalonic acid (MVA) pathway in both mitochondria and cytoplasm. A farnesyl pyrophosphate synthase mutant ERG20F96W/N127W and a linalool synthase from Cinnamomum osmophloeum (CoLIS) were introduced and meanwhile the endogenous ERG20 was down-regulated to prevent the competitive loss of precursor. In addition, overexpression of the proteins of CoLIS and ERG20F96W/N127W and another copy of the same enzymes CoLIS/ERG20F96W/N127W with mitochondrial localization signal (MLS) were carried out to further pull the flux to linalool. Finally, a maximum linalool titer of 23.45 mg/L was obtained in a batch fermentation with sucrose as carbon source. This combinatorial engineering strategy may provide hints for biosynthesis of other monoterpenes.

Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes

Pentacyclic triterpenes are diverse plant secondary metabolites derived from the mevalonate (MVA) pathway. Many of these molecules are potentially valuable, particularly as pharmaceuticals, and research has focused on their production in simpler and more amenable heterologous systems such as the yeast Saccharomyces cerevisiae. We have developed a new heterologous platform for the production of pentacyclic triterpenes in S. cerevisiae based on a combinatorial engineering strategy involving the overexpression of MVA pathway genes, the knockout of negative regulators, and the suppression of a competing pathway. Accordingly, we overexpressed S. cerevisiae ERG13, encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, and a truncated and deregulated variant of the rate-limiting enzyme HMG-CoA reductase 1 (tHMGR). In the same engineering step, we deleted the ROX1 gene, encoding a negative regulator of the MVA pathway and sterol biosynthesis, resulting in a push-and-pull strategy to enhance metabolic flux through the system. In a second step, we redirected this enhanced metabolic flux from late sterol biosynthesis to the production of 2,3-oxidosqualene, the direct precursor of pentacyclic triterpenes. In yeast cells transformed with a newly isolated sequence encoding lupeol synthase from the Russian dandelion (Taraxacum koksaghyz), we increased the yield of pentacyclic triterpenes by 127-fold and detected not only high levels of lupeol but also a second valuable pentacyclic triterpene product, β-amyrin.

Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane

An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP+ ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae

Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the PGAL1-controlled ISPS by overexpression of GAL 4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0mg/L to 23.6mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2mg/L of isoprene in sealed vials, and the isoprene production reached 640mg/L and 3.7g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals.

Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production

Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

An Active Enzyme Constructed from a 9-Amino Acid Alphabet

Nature employs a set of 20 amino acids to produce a repertoire of protein structures endowed with sophisticated functions. Here, we combined design and selection to create an enzyme composed entirely from a set of only 9 amino acids that can rescue auxotrophic cells lacking chorismate mutase. The simplified protein captures key structural features of its natural counterpart but appears to be somewhat less stable and more flexible. The potential of a dramatically reduced amino acid alphabet to produce an active catalyst supports the notion that primordial enzymes may have possessed low amino acid diversity and suggests that combinatorial engineering strategies, such as the one used here, may be generally applied to create enzymes with novel structures and functions.