Metabolic Engineering Strategies Research Articles

Coenzyme self-sufficiency system-recent advances in microbial production of high-value chemical phenyllactic acid.

Phenyllactic acid (PLA), a natural antimicrobial substance, has many potential applications in the food, animal feed, pharmaceutical and cosmetic industries. However, its production is limited by the complex reaction steps involved in its chemical synthesis. Through advances in metabolic engineering and synthetic biology strategies, enzymatic or whole-cell catalysis was developed as an alternative method for PLA production. Herein, we review recent developments in metabolic engineering and synthetic biology strategies that promote the microbial production of high-value PLA. Specially, the advantages and disadvantages of the using of the three kinds of substrates, which includes phenylpyruvate, phenylalanine and glucose as starting materials by natural or engineered microbes is summarized. Notably, the bio-conversion of PLA often requires the consumption of expensive coenzyme NADH. To overcome the issues of NADH regeneration, efficiently internal cofactor regeneration systems constructed by co-expressing different enzyme combinations composed of lactate dehydrogenase with others for enhancing the PLA production, as well as their possible improvements, are discussed. In particular, the construction of fusion proteins with different linkers can achieve higher PLA yield and more efficient cofactor regeneration than that of multi-enzyme co-expression. Overall, this review provides a comprehensive overview of PLA biosynthesis pathways and strategies for increasing PLA yield through biotechnology, providing future directions for the large-scale commercial production of PLA and the expansion of downstream applications.

Engineering the glyoxylate cycle for chemical bioproduction.

With growing concerns about environmental issues and sustainable economy, bioproduction of chemicals utilizing microbial cell factories provides an eco-friendly alternative to current petro-based processes. Creating high-performance strains (with high titer, yield, and productivity) through metabolic engineering strategies is critical for cost-competitive production. Commonly, it is inevitable to fine-tuning or rewire the endogenous or heterologous pathways in such processes. As an important pathway involved in the synthesis of many kinds of chemicals, the potential of the glyoxylate cycle in metabolic engineering has been studied extensively these years. Here, we review the metabolic regulation of the glyoxylate cycle and summarize recent achievements in microbial production of chemicals through tuning of the glyoxylate cycle, with a focus on studies implemented in model microorganisms. Also, future prospects for bioproduction of glyoxylate cycle-related chemicals are discussed.

Microbial detoxification of lignocellulosic biomass hydrolysates: Biochemical and molecular aspects, challenges, exploits and future perspectives.

Valorization of lignocellulosic biomass (LB) has the potential to secure sustainable energy production without impacting food insecurity, whist relieving over reliance on finite fossil fuels. Agro-derived lignocellulosic residues such as wheat straw, switchgrass, rice bran, and miscanthus have gained relevance as feedstocks for the production of biofuels and chemicals. However, the microorganisms employed in fermentative conversion of carbohydrates to fuels and chemicals are unable to efficiently utilize the sugars derived from LB due to co-production of lignocellulose-derived microbial inhibitory compounds (LDMICs) during LB pretreatment. LDMICs impact microbial growth by inhibition of specific enzymes, cause DNA and cell membrane damage, and elicit cellular redox imbalance. Over the past decade, success has been achieved with the removal of LDMICs prior to fermentation. However, LDMICs removal by chemical processes is often accompanied by sugar losses, which negatively impacts the overall production cost. Hence, in situ removal of LDMICs by fermentative organisms during the fermentation process has garnered considerable attention as the "go-to" approach for economical LDMICs detoxification and bio-chemicals production. In situ removal of LDMICs has been pursued by either engineering more robust biocatalysts or isolating novel microbial strains with the inherent capacity to mineralize or detoxify LDMICs to less toxic compounds. While some success has been made along this line, efficient detoxification and robust production of target bio-chemicals in lignocellulosic hydrolysates (LHs) under largely anaerobic fermentative conditions remains a lingering challenge. Consequently, LB remains an underutilized substrate for bio-chemicals production. In this review, the impact of microbial LH detoxification on overall target molecule production is discussed. Further, the biochemical pathways and mechanisms employed for in situ microbial detoxification of furanic LDMICs [e.g., furfural and 5-hydroxymethylfurfural (HMF)] and phenolic LDMICs (e.g., syringaldehyde, p-coumaric acid, 4-hydroxybenzaldehyde, vanillin, and ferulic acid) are discussed. More importantly, metabolic engineering strategies for the development of LDMIC-tolerant and bio-chemicals overproducing strains and processes are highlighted.

Nonconventional Yeasts Engineered Using the CRISPR-Cas System as Emerging Microbial Cell Factories

Because the petroleum-based chemical synthesis of industrial products causes serious environmental and societal issues, biotechnological production using microorganisms is an alternative approach to achieve a more sustainable economy. In particular, the yeast Saccharomyces cerevisiae is widely used as a microbial cell factory to produce biofuels and valuable biomaterials. However, product profiles are often restricted due to the Crabtree-positive nature of S. cerevisiae, and ethanol production from lignocellulose is possibly enhanced by developing alternative stress-resistant microbial platforms. With desirable metabolic pathways and regulation in addition to strong resistance to diverse stress factors, nonconventional yeasts (NCY) may be considered an alternative microbial platform for industrial uses. Irrespective of their high industrial value, the lack of genetic information and useful gene editing tools makes it challenging to develop metabolic engineering-guided scaled-up applications using yeasts. The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system is a powerful gene editing tool for NCYs. This review describes the current status of and recent advances in promising NCYs in terms of industrial and biotechnological applications, highlighting CRISPR-Cas9 system-based metabolic engineering strategies. This will serve as a basis for the development of novel yeast applications.

Mechanistic modeling of redox balance effects on the fermentation of eucalyptus wood-derived xylose to acetone-butanol-ethanol

A Michaelis-Menten-like kinetic model is proposed to simulate the effects of redox balance on Clostridium beijerinckii NCIMB 8052 metabolism during fermentation of biomass-derived xylose to acetone-butanol-ethanol (ABE). The model builds on previous metabolic kinetic models by introducing six mass balance differential equations for the redox pairs NADH/NAD+, NADPH/NADP+, and oxidized/reduced ferredoxin. Batch cultures were conducted using laboratory-grade or eucalyptus-derived xylose, and kinetic parameters were estimated by minimizing the difference between experimental (substrate, metabolites, and cell) and model results using a genetic optimization algorithm. The simulations agreed well with experimental data, and a sensitivity analysis showed that the rate of reoxidation of reduced ferredoxin used for NADPH regeneration is the most relevant reaction for the synthesis of acids and ABE. Furthermore, an increase in NADPH consumption due to the reduction of eucalyptus-derived furfural to furfuryl alcohol was only significant for the production of butanol. Differences in the values of the Michaelis-Menten constants, Km’s, depending on the xylose source, suggest that a decrease in the conversion rates of lactate to pyruvate and acetate to acetyl-CoA considerably affected the biosynthesis of butanol from eucalyptus-derived xylose. The proposed model was effective in assessing the effects of redox balance on ABE fermentation, suggesting its use for the design of metabolic engineering strategies to improve butanol synthesis from furfural-containing biomass hydrolysates.

The De Novo Synthesis of 2-Phenylethanol from Glucose by the Synthetic Microbial Consortium Composed of Engineered Escherichia coli and Meyerozyma guilliermondii.

Synthetic microbial consortia show promising applications for fine chemical production, especially with long metabolic pathways. In this study, a synthetic microbial consortium consisting of Escherichia coli YLC20 and Meyerozyma guilliermondii MG57 was successfully constructed, which could achieve efficient de novo 2-phenylethanol (2-PE) production from glucose. A tyrosine-deficient E.coli YLC20 overexpressing genes of aroF and pheA was first constructed, which could accumulate 29.5 g/L of l-phenylalanine (l-Phe) within 96 h from glucose accompanied by the coproduction of acetate and α-ketoglutarate (α-KG). Furthermore, the engineered M.guilliermondii MG57 was constructed through the stepwise metabolic engineering strategy, which could facilitate the 2-PE synthesis from l-Phe. Moreover, the cosubstrate and material intervention strategies were applied to improve the stability of the microbial consortium and 2-PE production. Finally, the synthetic microbial consortium could de novo synthesize 3.77 g/L of 2-PE from 80 g/L of glucose, providing a reference for the de novo synthesis of fine chemicals with long metabolic pathways.

Nutrient-Limited Operational Strategies for the Microbial Production of Biochemicals.

Limiting an essential nutrient has a profound impact on microbial growth. The notion of growth under limited conditions was first described using simple Monod kinetics proposed in the 1940s. Different operational modes (chemostat, fed-batch processes) were soon developed to address questions related to microbial physiology and cell maintenance and to enhance product formation. With more recent developments of metabolic engineering and systems biology, as well as high-throughput approaches, the focus of current engineers and applied microbiologists has shifted from these fundamental biochemical processes. This review draws attention again to nutrient-limited processes. Indeed, the sophisticated gene editing tools not available to pioneers offer the prospect of metabolic engineering strategies which leverage nutrient limited processes. Thus, nutrient- limited processes continue to be very relevant to generate microbially derived biochemicals.

De novo transcriptome profiling unveils the regulation of phenylpropanoid biosynthesis in unripe Piper nigrum berries

BackgroundBlack pepper (Piper nigrum L.) is rich in bioactive compounds that make it an imperative constituent in traditional medicines. Although the unripe fruits have long been used in different Ayurvedic formulations, the mechanism of gene regulation resulting in the production of the bioactive compounds in black pepper is not much investigated. Exploring the regulatory factors favouring the production of bioactive compounds ultimately help to accumulate the medicinally important content of black pepper. The factors that enhance the biosynthesis of these compounds could be potential candidates for metabolic engineering strategies to obtain a high level production of significant biomolecules.ResultsBeing a non-model plant, de novo sequencing technology was used to unravel comprehensive information about the genes and transcription factors that are expressed in mature unripe green berries of P. nigrum from which commercially available black pepper is prepared. In this study, the key gene regulations involved in the synthesis of bioactive principles in black pepper was brought out with a focus on the highly expressed phenylpropanoid pathway genes. Quantitative real-time PCR analysis of critical genes and transcription factors in the different developmental stages from bud to the mature green berries provides important information useful for choosing the developmental stage that would be best for the production of a particular bioactive compound. Comparison with a previous study has also been included to understand the relative position of the results obtained from this study.ConclusionsThe current study uncovered significant information regarding the gene expression and regulation responsible for the bioactivity of black pepper. The key transcription factors and enzymes analyzed in this study are promising targets for achieving a high level production of significant biomolecules through metabolic engineering.

β-carotene genetically-enriched lyophilized orange juice increases antioxidant capacity and reduces β-amyloid proteotoxicity and fat accumulation in Caenorhabditis elegans

Citrus sinensis orange juice is an excellent dietary source of β-carotene, a well-known antioxidant. However, β-carotene concentrations are relatively low in most cultivars. We developed a new orange through metabolic engineering strategy (GS) with 33.72-fold increase in β-carotene content compared to its conventional counterpart (CV). Using Caenorhabditis elegans, we found that animals treated with GS showed a greater reduction in intracellular reactive oxygen species (ROS) which is associated with a greater resistance to oxidative stress and induction of the expression of antioxidant genes. Moreover, animals treated with GS orange showed a more effective protection against β-amyloid proteotoxicity and greater hypolipidemic effect under high glucose diet compared to animals treated with CV. These data demonstrate that the increased amount of β-carotene in orange actually provides a greater beneficial effect in C. elegans and a valuable proof of principle to support further studies in mammals and humans.

An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis.ResultsConsistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD+-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose–sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high Km for CO2 of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO2. Prolonged chemostat cultivation on glucose–sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway.ConclusionsCombination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.

β-Farnesene Production from Low-Cost Glucose in Lignocellulosic Hydrolysate by Engineered Yarrowia lipolytica

β-Farnesene is value-added acyclic volatile sesquiterpene with wide applications in energy, industry, and agriculture. Producing high-value-added compounds from low-cost renewable feedstocks in engineered microbial cell factories is an environmentally friendly and economical process for β-farnesene biosynthesis. In this study, the potential for using engineered Yarrowia lipolytica to produce β-farnesene from lignocellulosic hydrolysate as the carbon source was investigated. An efficient biosynthetic pathway for β-farnesene production was established via iterative enhancement of multiple genes based on the high endogenous acetyl-CoA flux in Yarrowia lipolytica. Overexpression of mevalonate pathway genes and screening of β-farnesene synthase resulted in a β-farnesene titer of 245 mg L−1 in glucose media. Additional copies of mevalonate pathway genes and enhanced expression of HMG-CoA reductase and β-farnesene synthase further increased the titer of β-farnesene to 470 mg L−1. In addition, by combining metabolic engineering strategies using the lignocellulosic hydrolysate utilization strategy, the addition of Mg2+ promoted the production of β-farnesene, and the best-performing strain produced 7.38 ± 0.24 g L−1 β-farnesene from lignocellulosic hydrolysate media in a 2 L fermenter after 144 h. This study shows great potential for the sustainable production of β-farnesene from lignocellulosic biomass via engineered Yarrowia lipolytica.

Enhancing microalgal lipid accumulation for biofuel production.

Microalgae have high lipid accumulation capacity, high growth rate and high photosynthetic efficiency which are considered as one of the most promising alternative sustainable feedstocks for producing lipid-based biofuels. However, commercialization feasibility of microalgal biofuel production is still conditioned to the high production cost. Enhancement of lipid accumulation in microalgae play a significant role in boosting the economics of biofuel production based on microalgal lipid. The major challenge of enhancing microalgal lipid accumulation lies in overcoming the trade-off between microalgal cell growth and lipid accumulation. Substantial approaches including genetic modifications of microalgal strains by metabolic engineering and process regulations of microalgae cultivation by integrating multiple optimization strategies widely applied in industrial microbiology have been investigated. In the present review, we critically discuss recent trends in the application of multiple molecular strategies to construct high performance microalgal strains by metabolic engineering and synergistic strategies of process optimization and stress operation to enhance microalgal lipid accumulation for biofuel production. Additionally, this review aims to emphasize the opportunities and challenges regarding scaled application of the strategic integration and its viability to make microalgal biofuel production a commercial reality in the near future.

Metabolic engineering enables Bacillus licheniformis to grow on the marine polysaccharide ulvan

BackgroundMarine algae are responsible for half of the global primary production, converting carbon dioxide into organic compounds like carbohydrates. Particularly in eutrophic waters, they can grow into massive algal blooms. This polysaccharide rich biomass represents a cheap and abundant renewable carbon source. In nature, the diverse group of polysaccharides is decomposed by highly specialized microbial catabolic systems. We elucidated the complete degradation pathway of the green algae-specific polysaccharide ulvan in previous studies using a toolbox of enzymes discovered in the marine flavobacterium Formosa agariphila and recombinantly expressed in Escherichia coli.ResultsIn this study we show that ulvan from algal biomass can be used as feedstock for a biotechnological production strain using recombinantly expressed carbohydrate-active enzymes. We demonstrate that Bacillus licheniformis is able to grow on ulvan-derived xylose-containing oligosaccharides. Comparative growth experiments with different ulvan hydrolysates and physiological proteogenomic analyses indicated that analogues of the F. agariphila ulvan lyase and an unsaturated β-glucuronylhydrolase are missing in B. licheniformis. We reveal that the heterologous expression of these two marine enzymes in B. licheniformis enables an efficient conversion of the algal polysaccharide ulvan as carbon and energy source.ConclusionOur data demonstrate the physiological capability of the industrially relevant bacterium B. licheniformis to grow on ulvan. We present a metabolic engineering strategy to enable ulvan-based biorefinery processes using this bacterial cell factory. With this study, we provide a stepping stone for the development of future bioprocesses with Bacillus using the abundant marine renewable carbon source ulvan.

Combinatorial Metabolic Engineering Strategies for the Enhanced Production of Free Fatty Acids in Escherichia coli.

In this study, we evaluated the effects of several metabolic engineering strategies in a systematic and combinatorial manner to enhance the free fatty acid (FFA) production in Escherichia coli. The strategies included (i) overexpression of mutant thioesterase I ('TesAR64C) to efficiently release the FFAs from fatty acyl-ACP; (ii) coexpression of global regulatory protein FadR; (iii) heterologous expression of methylmalonyl-CoA carboxyltransferase and phosphoenolpyruvate carboxylase to synthesize fatty acid precursor molecule malonyl-CoA; and (iv) disruption of genes associated with membrane proteins (GusC, MdlA, and EnvR) to improve the cellular state and export the FFAs outside the cell. The synergistic effects of these genetic modifications in strain SBF50 yielded 7.2 ± 0.11 g/L FFAs at the shake flask level. In fed-batch cultivation under nitrogen-limiting conditions, strain SBF50 produced 33.6 ± 0.02 g/L FFAs with a productivity of 0.7 g/L/h from glucose, which is the maximum titer reported in E. coli to date. Combinatorial metabolic engineering approaches can prove to be highly useful for the large-scale production of FA-derived chemicals and fuels.

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor.

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.

Construction and yield optimization of a cinnamylamine biosynthesis route in Escherichia coli

BackgroundWith the development of metabolic engineering and synthetic biology, the biosynthesis of aromatic compounds has attracted much attention. Cinnamylamine is an aromatic compound derived from l-phenylalanine, which is used in the synthesis of biologically active molecules, including drugs, and energetic materials. Cinnamylamine has been mainly synthesized by chemical methods to date, and few reports have focused on the biosynthesis of cinnamylamine. Therefore, it is desirable to establish an efficient biosynthesis method for cinnamylamine.ResultsThe ω-aminotransferase Cv-ωTA from Chromobacterium violaceum has been demonstrated to have high enzyme activity in the conversion of cinnamaldehyde to cinnamylamine. To prevent the preferable conversion of cinnamaldehyde to cinnamyl alcohol in wild-type Escherichia coli, the E. coli MG1655 strain with reduced aromatic aldehyde reduction (RARE) in which six aldehyde ketone reductase and alcohol dehydrogenase genes have been knocked out was employed. Then, the carboxylic acid reductase from Neurospora crassa (NcCAR) and phosphopantetheinyl transferase (PPTase) from E. coli were screened for a high conversion rate of cinnamic acid to cinnamaldehyde. To shift the equilibrium of the reaction toward cinnamylamine, saturation mutagenesis of Cv-ωTA at key amino acid residues was performed, and Cv-ωTA Y168G had the highest conversion rate with 88.56 mg/L cinnamylamine obtained after 4 h of fermentation. Finally, by optimizing the substrates and the supply of the cofactors, PLP and NADPH, in the fermentation, the yield of cinnamylamine in engineered E. coli reached 523.15 mg/L.ConclusionWe achieved the first biosynthesis of cinnamylamine using cinnamic acid as the precursor in E. coli using a combinatorial metabolic engineering strategy. This study provides a reference for the biosynthesis of other amine compounds and lays a foundation for the de novo synthesis of cinnamylamine.

Bioprocess Engineering, Transcriptome, and Intermediate Metabolite Analysis of L-Serine High-Yielding Escherichia coli W3110.

L-serine is widely used in the food, cosmetic, and pharmaceutical industries. However, the complicated metabolic network and regulatory mechanism of L-serine production lead to the suboptimal productivity of the direct fermentation of L-serine and limits its large-scale industrial production. In this study, a high-yield L-serine production Escherichia coli strain was constructed by a series of defined genetic modification methodologies. First, L-serine-mediated feedback inhibition was removed and L-serine biosynthetic pathway genes (serAfr, serC, and serB) associated with phosphoglycerate kinase (pgk) were overexpressed. Second, the L-serine conversion pathway was further examined by introducing a glyA mutation (K229G) and deleting other degrading enzymes based on the deletion of initial sdaA. Finally, the L-serine transport system was rationally engineered to reduce uptake and accelerate L-serine export. The optimally engineered strain produced 35 g/L L-serine with a productivity of 0.98 g/L/h and a yield of 0.42 g/g glucose in a 5-L fermenter, the highest productivity and yield of L-serine from glucose reported to date. Furthermore, transcriptome and intermediate metabolite of the high-yield L-serine production Escherichia coli strain were analyzed. The results demonstrated the regulatory mechanism of L-serine production is delicate, and that combined metabolic and bioprocess engineering strategies for L-serine producing strains can improve the productivity and yield.

The Advancements and Prospects of Nervonic Acid Production.

Nervonic acid (NA) is a monounsaturated very long-chain fatty acid (VLCFA) and has been identified with critical biological functions in medical and health care for brain development and injury repair. Yet, the approaches to producing NA from the sources of plants or animals continue to pose challenges to meet increasing market demand, as they are generally associated with high costs, a lack of natural resources, a long life cycle, and low production efficiency. The recent technological advance in metabolic engineering allows us to precisely engineer oleaginous microbes to develop high-content NA-producing strains, which has the potential to provide a possible solution to produce NA on a commercial fermentation scale. In this Review, the biosynthetic pathway, natural sources, and metabolic engineering of NA are summarized. The strategies of metabolic engineering that could be adopted to modify oleaginous yeast to produce NA are discussed in detail, providing the prospecting views for the microbial cells producing NA.

Glycosyltransferase engineering and multi-glycosylation routes development facilitating synthesis of high-intensity sweetener mogrosides

SummaryMogrosides are widely served as natural zero-calorie sweeteners. To date, the biosynthesis of high-intensity sweetness mogrosides V from mogrol has not been achieved because of inefficient and uncontrollable multi-glycosylation process. To address this challenge, we reported three UDP-glycosyltransferases (UGTs) catalyzing the primary and branched glycosylation of mogrosides and increased the catalytic efficiency by 74–400-folds toward branched glycosylation using an activity-based sequence conservative analysis engineering strategy. The computational studies provided insights into the origin of improved catalytic activity. By virtue of UGT mutants, we provided regio- and bond-controllable multi-glycosylation routes, successfully facilitating sequential glycosylation of mogrol to three kinds of mogroside V in excellent yield of 91–99%. Meanwhile, the feasibility of the routes was confirmed in engineered yeasts. It suggested that the multi-glycosylation routes would be combined with mogrol synthetic pathway to de novo produce mogrosides from glucose by aid of metabolic engineering and synthetic biology strategies in the future.

Multi-level metabolic engineering of Escherichia coli for high-titer biosynthesis of 2'-fucosyllactose and 3-fucosyllactose.

Fucosyllactoses (FL), including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high-titer FL biosynthesis by introducing multi-level metabolic engineering strategies, including (1) individual construction of the 2'/3-FL-producing strains through gene combination optimization of the GDP-L-fucose module; (2) screening of rate-limiting enzymes (α-1,2-fucosyltransferase and α-1,3-fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate-limiting enzymes by the RBS screening, fusion peptides and multi-copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2'-FL and 6.28 g/L for 3-FL in shake flasks with a modified-M9CA medium. Fed-batch cultivations of the two strains generated 64.62 g/L of 2'-FL and 40.68 g/L of 3-FL in the 3-L bioreactors, with yields of 0.65 mol 2'-FL/mol lactose and 0.67 mol 3-FL/mol lactose, respectively. This research provides a viable platform for other high-value-added compounds production in microbial cell factories.