Microbial Metabolic Engineering Research Articles

Insights into the Structures, Inhibitors, and Improvement Strategies of Glucose Oxidase

Glucose oxidase, which uses molecular oxygen as an electron acceptor to specifically catalyze the conversion of β-d-glucose to gluconic acid and hydrogen peroxide (H2O2), has been considered an important enzyme in increasing environmental sustainability and food security. However, achieving the high yield, low price and high activity required for commercial viability remains challenging. In this review, we first present a brief introduction, looking at the sources, characteristics, catalytic process, and applications of glucose oxidase. Then, the predictive structures of glucose oxidase from two different sources are comparatively discussed. We summarize the inhibitors of glucose oxidase. Finally, we highlight how the production of glucose oxidase can be improved by optimizing the culture conditions and microbial metabolic engineering.

Opportunities in the microbial valorization of sugar industrial organic waste to biodegradable smart food packaging materials

Many petroleum-derived plastics, including food packaging materials are non-biodegradable and designed for single-use applications. Annually, around 175 Mt. of plastic enters the land and ocean ecosystems due to mismanagement and lack of techno economically feasible plastic waste recycling technologies. Renewable sourced, biodegradable polymer-based food packaging materials can reduce this environmental pollution. Sugar production from sugarcane or sugar beet generates organic waste streams that contain fermentable substrates, including sugars, acids, and aromatics. Microbial metabolism can be leveraged to funnel those molecules to platform chemicals or biopolymers to generate biodegradable food packaging materials that have active or sensing molecules embedded in biopolymer matrices. The smart package can real-time monitor food quality, assure health safety, and provide economic and environmental benefits. Active packaging materials display functional properties such as antimicrobial, antioxidant, and light or gas barrier. This article provides an overview of potential biodegradable smart/active polymer packages for food applications by valorizing sugar industry-generated organic waste. We highlight the potential microbial pathways and metabolic engineering strategies to biofunnel the waste carbon efficiently into the targeted platform chemicals such as lactic, succinate, muconate, and biopolymers, including polyhydroxyalkanoates, and bacterial cellulose. The obtained platform chemicals can be used to produce biodegradable polymers such as poly (butylene adipate-co-terephthalate) (PBAT) that could replace incumbent polyethylene and polypropylene food packaging materials. When nanomaterials are added, these polymers can be active/smart. The process can remarkably lower the greenhouse gas emission and energy used to produce food-packaging material via sugar industrial waste carbon relative to the petroleum-based production. The proposed green routes enable the valorization of sugar processing organic waste into biodegradable materials and enable the circular economy.

Metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3β,20S-Di-O-Glc-DM

Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity. Traditional approaches for producing them rely on direct extraction from Panax ginseng, enzymatic catalysis or chemical synthesis, all of which result in low yield and high cost. Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products. Hence we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-O-Glc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titers of F2 and 3β,20S-Di-O-Glc-DM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level, respectively, by multistep metabolic engineering strategies. Additionally, pharmacological evaluation showed that both F2 and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-Glc-DM was even better. Furthermore, the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor. To our knowledge, this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM, and achieving their de novo biosynthesis by the engineered yeasts. Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass, which lays a foundation for drug research and development.

Identification and Characterization of Two Regiospecific Tricetin UDP-Dependent Glycosyltransferases from Pomegranate (Punica granatum L.).

Tricetin (5,7,3′,4′,5′-pentahydroxyflavone) is a dietary flavone from flowers of Myrtales plants with demonstrated functions in promoting human health. By contrast, the bioactivity of its glucosylated derivative tricetin 4′-O-glucoside has not been extensively explored. We conducted metabolite profiling analysis of pomegranate (a Myrtales plant) floral tissues and revealed that tricetin and tricetin 4′-O-glucoside accumulate in anthers, but not petals. In addition, the comparative analysis of anther and petal transcriptomes identified 10 UGTs that are more highly expressed in anthers than petals. Of the 10 UGTs, PgUGT76Z1 and PgUGT73AL1 glucosylated specifically at the 4′-O position of tricetin to form tricetin 4′-O-glucoside. The phylogenetic analysis indicated that PgUGT76Z1 and PgUGT73AL1 belong to different plant UGT groups, suggesting a convergent evolution of these tricetin UGTs. Overall, identification and characterization of PgUGT76Z1 and PgUGT73AL1 not only provides evolutionary insights into tricetin glucosylation, but also offers an opportunity to produce tricetin 4′-O-glucoside in large quantities through microbial biotransformation or plant metabolic engineering, thus facilitating the investigation of tricetin 4′-O-glucoside bioactivities.

Bacterial bioactive metabolites as therapeutic agents: From production to action

Bacterial metabolites are one of the primary sources of drugs that we currently use to treat several diseases. However, bacterial drug discovery and development is a challenging and time-consuming process, and the emergence of new diseases and the development of resistance to currently available drugs demand the discovery of new metabolites with better biological activities. The new advancements in microbial technology, omics, genome and metabolic engineering, synthetic biology and the interdisciplinary approach of these fields overcome the hurdles in drug discovery and heterologous synthesis from bacteria. The gut microbiome performs a vital role in sustaining human health and aids in tackling various diseases. The metabolites produced by the gut microbiome act as an energy source for colon epithelium, maintain pH, help in cell differentiation and induces apoptosis in abnormal cells. The review discusses about the bacterial derived bioactive compounds, advancements and technologies in bacterial synthesis of bioactive sources and genomic and synthetic biology methods for the bioprospecting of bacterial metabolites. Since the gut microbiome relates to colon health, we have also discussed the techniques comprising probiotics, prebiotics, microbiome transplantation, toxins, and bacteriocins capable of preventing and managing colon associated health condition. Future directions in bacterial bioactive metabolite production are also discussed.

Advances in microbial metabolic engineering for the production of butanol isomers (isobutanol and 1-butanol) from a various biomass

Advances in microbial metabolic engineering for the production of butanol isomers (isobutanol and 1-butanol) from a various biomass

Engineering eukaryote-like regulatory circuits to expand artificial control mechanisms for metabolic engineering in Saccharomyces cerevisiae

Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast (Saccharomyces cerevisiae) suffers from several drawbacks; specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae. Trans- and cis- modules, including eukaryotic trans-activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L−1 in flask cultivation. These negative/positive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.

Metabolic engineering study on biosynthesis of 4-hydroxybenzyl alcohol from L-tyrosine in Escherichia coli

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.

Chromosome-level assembly of the Neolamarckia cadamba genome provides insights into the evolution of cadambine biosynthesis.

Neolamarckia cadamba (Roxb.), a close relative of Coffea canephora and Ophiorrhiza pumila, is an important traditional medicine in Southeast Asia. Three major glycosidic monoterpenoid indole alkaloids (MIAs), cadambine and its derivatives 3β-isodihydrocadambine and 3β-dihydrocadambine, accumulate in the bark and leaves, and exhibit antimalarial, antiproliferative, antioxidant, anticancer and anti-inflammatory activities. Here, we report a chromosome-scale N. cadamba genome, with 744.5 Mb assembled into 22 pseudochromosomes with contig N50 and scaffold N50 of 824.14 Kb and 29.20 Mb, respectively. Comparative genomic analysis of N. cadamba with Co. canephora revealed that N. cadamba underwent a relatively recent whole-genome duplication (WGD) event after diverging from Co. canephora, which contributed to the evolution of the MIA biosynthetic pathway. We determined the key intermediates of the cadambine biosynthetic pathway and further showed that NcSTR1 catalyzed the synthesis of strictosidine in N. cadamba. A new component, epoxystrictosidine (C27H34N2O10, m/z 547.2285), was identified in the cadambine biosynthetic pathway. Combining genome-wide association study (GWAS), population analysis, multi-omics analysis and metabolic gene cluster prediction, this study will shed light on the evolution of MIA biosynthetic pathway genes. This N. cadamba reference sequence will accelerate the understanding of the evolutionary history of specific metabolic pathways and facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants.

Physiological limitations and opportunities in microbial metabolic engineering.

Metabolic engineering can have a pivotal role in increasing the environmental sustainability of the transportation and chemical manufacturing sectors. The field has already developed engineered microorganisms that are currently being used in industrial-scale processes. However, it is often challenging to achieve the titres, yields and productivities required for commercial viability. The efficiency of microbial chemical production is usually dependent on the physiological traits of the host organism, which may either impose limitations on engineered biosynthetic pathways or, conversely, boost their performance. In this Review, we discuss different aspects of microbial physiology that often create obstacles for metabolic engineering, and present solutions to overcome them. We also describe various instances in which natural or engineered physiological traits in host organisms have been harnessed to benefit engineered metabolic pathways for chemical production.

Reprogramming Escherichia coli pyruvate-forming reaction towards chorismate derivatives production

Microbial metabolic pathway engineering is a potent strategy used worldwide to produce aromatic compounds. We drastically rewired the primary metabolic pathway of Escherichia coli to produce aromatics and their derivatives. The metabolic pathway of E. coli was compartmentalized into the production and energy modules. We focused on the pyruvate-forming reaction in the biosynthesis pathway of some compounds as the reaction connecting those modules. E. coli strains were engineered to show no growth unless pyruvate was synthesized along with the compounds of interest production. Production of salicylate and maleate was demonstrated to confirm our strategy's versatility. In maleate production, the production, yield against the theoretical yield, and production rate reached 12.0 g L−1, 67%, and up to fourfold compared to that in previous reports, respectively; these are the highest values of maleate production in microbes to our knowledge. The results reveal that our strategy strongly promotes the production of aromatics and their derivatives.

Application of CRISPR/Cas System in the Metabolic Engineering of Small Molecules

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas protein technology area is rapidly growing technique for genome editing and modulation of transcription of several microbes. Successful engineering in microbes requires an emphasis on the aspect of efficiency and targeted aiming, which can be employed using CRISPR/Cas system. Hence, this type of system is used to modify the genome of several microbes such as yeast and bacteria. In recent years, CRISPR/Cas systems have been chosen for metabolic engineering in microbes due to their specificity, orthogonality, and efficacy. Therefore, we need to review the scheme which was acquired for the execution of the CRISPR/Cas system for the modification and metabolic engineering in yeast and bacteria. In this review, we highlighted the application of the CRISPR/Cas system which has been used for the production of small molecules in the microbial system that is chemically and biologically important.

Development and high yield strategies of microbial-derived antibiotics in agriculture

The development of high-efficiency, low-toxicity, and low-residue green pesticides is the main trend of pesticide research, and the microbial-derived antibiotics are one of the essential parts of green pesticides and play a significant role in agriculture. With the development of microbial genomics technology, metabolic engineering, high-throughput screening and other technologies, the research on new microbial-derived antibiotics has entered a new stage in agriculture. Here we briefly summarize the types of new microbial-derived antibiotics developed in agriculture over the past decade. We also introduce the research strategies for high-yield breeding and fermentation of antibiotic-producing strains in agriculture. This review may provide references for the future development of agricultural antibiotics.

Erythritol Chemical Structure, Biosynthesis Pathways, Properties, Applications, and Production

Erythritol is a natural four carbon sugar alcohol present in fruits, vegetables, mushrooms and in fermented foods. It is low calorie sweetener manufactured commercially by fermentation using osmophilic yeasts and yeasts-like fungi. Some filamentous fungi and heterofermentative lactic acid bacteria demonstrated on laboratory scale the production of erythritol at lower yield. Erythritol is gaining high market share as natural sweetener with applications in low calorie foods, beverages, and as an additive in combination with high intense zero calorie artificial sweeteners to enhance sweetness, texture, and to mask these artificial sweeteners bitter after taste. Due to increasing demand of erythritol with wide applications in foods, pharmaceuticals, and chemical industries, research activities are focusing on reducing production cost by fermentation conditions optimization. and improve industrial cultures via mutation or genetic engineering to utilize cheap and abundant byproducts such as crude glycerol and lignocellulosic materials as carbon sources in replacement to the costly glucose. Erythritol production with less intermediate metabolites in the fermentation process can be achieved by microbial metabolic pathway engineering. High yield of erythritol with less intermediate metabolites at the end of fermentation process will lead to improve erythritol recovery efficiency with higher yield and specifications.

Microbial Upgrading of Acetate into Value-Added Products-Examining Microbial Diversity, Bioenergetic Constraints and Metabolic Engineering Approaches.

Ecological concerns have recently led to the increasing trend to upgrade carbon contained in waste streams into valuable chemicals. One of these components is acetate. Its microbial upgrading is possible in various species, with Escherichia coli being the best-studied. Several chemicals derived from acetate have already been successfully produced in E. coli on a laboratory scale, including acetone, itaconic acid, mevalonate, and tyrosine. As acetate is a carbon source with a low energy content compared to glucose or glycerol, energy- and redox-balancing plays an important role in acetate-based growth and production. In addition to the energetic challenges, acetate has an inhibitory effect on microorganisms, reducing growth rates, and limiting product concentrations. Moreover, extensive metabolic engineering is necessary to obtain a broad range of acetate-based products. In this review, we illustrate some of the necessary energetic considerations to establish robust production processes by presenting calculations of maximum theoretical product and carbon yields. Moreover, different strategies to deal with energetic and metabolic challenges are presented. Finally, we summarize ways to alleviate acetate toxicity and give an overview of process engineering measures that enable sustainable acetate-based production of value-added chemicals.

Synthetic small regulatory RNAs in microbial metabolic engineering.

Small regulatory RNAs (sRNAs) finely control gene expression in prokaryotes and synthetic sRNA has become a useful high-throughput approach to tackle current challenges in metabolic engineering because of its many advantages compared to conventional gene knockouts. In this review, we first focus on the modular structures of sRNAs and rational design strategies of synthetic sRNAs on the basis of their modular structures. The wide applications of synthetic sRNAs in bacterial metabolic engineering, with or without the aid of heterogeneously expressed Hfq protein, were also covered. In addition, we give attention to the improvements in implementing synthetic sRNAs, which make the synthetic sRNA strategy universally applicable in metabolic engineering and synthetic biology. KEY POINTS: • Synthetic sRNAs can be rationally designed based on modular structures of natural sRNAs. • Synthetic sRNAs were widely used for metabolic engineering in various microorganisms. • Several technological improvements made the synthetic sRNA strategy more applicable.

High level production of amorphadiene using Bacillus subtilis as an optimized terpenoid cell factory

The anti-malarial drug artemisinin, produced naturally in the plant Artemisia annua, experiences unstable and insufficient supply as its production relies heavily on the plant source. To meet the massive demand for this compound, metabolic engineering of microbes has been studied extensively. In this study, we focus on improving the production of amorphadiene, a crucial artemisinin precursor, in Bacillus subtilis. The expression level of the plant-derived amorphadiene synthase (ADS) was upregulated by fusion with green fluorescent protein (GFP). Furthermore, a co-expression system of ADS and a synthetic operon carrying the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes was established. Subsequently, farnesyl pyrophosphate synthase (FPPS), a key enzyme in formation of the sesquiterpene precursor farnesyl pyrophosphate (FPP), was expressed to supply sufficient substrate for ADS. The consecutive combination of these features yielded a B. subtilis strain expressing chromosomally integrated GFP-ADS followed by FPPS and a plasmid encoded synthetic operon showing a stepwise increased production of amorphadiene. An experimental design-aided systematic medium optimization was used to maximize the production level for the most promising engineered B. subtilis strain, resulting in an amorphadiene yield of 416 ± 15 mg/L, which is 20-fold higher than that previously reported in B. subtilis and more than double the production in Escherichia coli or Saccharomyces cerevisiae on a shake flask fermentation level.

Advances in Strain Engineering for Improved Bio-fuel Production- a Perspective

Bio-fuels are ecologically sustainable alternates of fossil fuel and have attracted interest of research community in the last few decades. Microorganisms such as bacteria, fungi and microalgae have important roles to play at various steps of bio-fuel production. And therefore several efforts such as genetic engineering have been made to improve the performance of these microbes to achieve the desired results. Metabolic engineering of organisms has benefitted immensely from the novel tools and technologies that have recently been developed. Microorganisms have the advantage of smaller and less complex genome and hence are best suitable for genetic manipulations. In this perspective, we briefly review a few interesting studies which represent some recent advances in the field of metabolic engineering of microbes.

Comparative Genomics of Spatholobus suberectus and Insight Into Flavonoid Biosynthesis.

Spatholobus suberectus Dunn (S. suberectus), has been widely used in traditional medicines plant source of the Leguminosae family. Its vine stem of which plays an important role in the prevention and treatment of various diseases because it contains various flavonoids. Comparative genome analysis suggested well-conserved genomic components and genetic collinearity between the genome of S. suberectus and other genera of Leguminosae such as Glycine max. We discovered two whole genome duplications (WGD) events in S. suberectus and G. max lineage underwent a WGD after speciation from S. suberectus. The determination of expansion and contractions of orthologous gene families revealed 1,001 expanded gene families and 3,649 contracted gene families in the S. suberectus lineage. Comparing to the model plants, many novel flavonoid biosynthesis-related genes were predicted in the genome of S. suberectus, and the expression patterns of these genes in the roots are similar to those in the stems [such as the isoflavone synthase (IFS) genes]. The expansion of IFS from a single copy in the Leguminosae ancestor to four copies in S. suberectus, will accelerate the biosynthesis of flavonoids. MYB genes are widely involved in plant flavonoid biosynthesis and the most abundant member of the TF family in S. suberectus. Activated retrotransponson positive regulates the accumulation of flavonoid in S. suberectus by introducing the cis-elements of tissue-specific expressed MYBs. Our study not only provides significant insight into the evolution of specific flavonoid biosynthetic pathways in S. suberectus, but also would facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding for alleviating resource shortage of S. suberectus.

From lab bench to formulated ingredient: Characterization, production, and commercialization of human milk oligosaccharides

Abstract Human milk oligosaccharides (HMOs) are known to positively influence infant health. Extensive variation exists in the levels, diversity, and complexity of oligosaccharides in the milk of a lactating mother. Until recently, limited availability of HMOs hampered their use in clinical applications. Most HMOs are unique to human milk, and have proven difficult and expensive to isolate and synthesize. Added to that, analysis of these complex glycans in milk samples requires state-of-the-art analytical instruments and associated technologies. The current review provides a critical overview of methods used in HMO analysis, and highlights the importance of understanding the factors which influence their composition and structural diversity. We also discuss recently employed strategies to overcome the availability of HMOs at industrial scale including microbial metabolic engineering and chemoenzymatic techniques. Finally, we examine how these recent advancements have opened up new avenues for future research and nutraceutical applications.