Metabolic Engineering and Synthetic Biology Approaches for the Heterologous Production of Aromatic Polyketides.

Polyketides are a diverse set of natural products with versatile applications as pharmaceuticals, nutraceuticals, and cosmetics, to name a few. Of several types of polyketides, aromatic polyketides comprising type II and III polyketides contain many chemicals important for human health such as antibiotics and anticancer agents. Most aromatic polyketides are produced from soil bacteria or plants, which are difficult to engineer and grow slowly in industrial settings. To this end, metabolic engineering and synthetic biology have been employed to efficiently engineer heterologous model microorganisms for enhanced production of important aromatic polyketides. In this review, we discuss the recent advancement in metabolic engineering and synthetic biology strategies for the production of type II and type III polyketides in model microorganisms. Future challenges and prospects of aromatic polyketide biosynthesis by synthetic biology and enzyme engineering approaches are also discussed.

Production of phenylpropanoids and flavonolignans from glycerol by metabolically engineered Escherichia coli.

Phenylpropanoids are a group of plant natural products with medicinal importance derived from aromatic amino acids. Here, we report the production of two representative phenylpropanoids-coniferyl alcohol (CA) and dihydroquercetin (DHQ)-from glycerol by engineered Escherichia coli. First, an E. coli strain capable of producing 187.7 mg/L of CA from glycerol was constructed by the introduction of hpaBC from E. coli and OMT1, 4CL4, and CCR1 from Arabidopsis thaliana to the p-coumaric acid producer. Next, an E. coli strain capable of producing 239.4 mg/L of DHQ from glycerol was constructed by the introduction of F3H, TT7, and CPR from A. thaliana to the naringenin producer, followed by engineering the signal peptide of a cytochrome P450 TT7. Furthermore, to demonstrate the production of flavonolignans, a group of heterodimeric phenylpropanoids, from glycerol, ascorbate peroxidase 1 from Silybum marianum was employed and engineered to produce 0.04 µg/L of silybin and 1.29 µg/L of isosilybin from glycerol by stepwise culture. Finally, a single strain harboring all the 16 necessary genes was constructed, resulting in 0.12 µg/L of isosilybin production directly from glycerol. The strategies described here will be useful for the production of pharmaceutically important yet complex natural products.

Application of Lactic Acid Bacteria (LAB) in Sustainable Agriculture: Advantages and Limitations.

Lactic acid bacteria (LAB) are significant groups of probiotic organisms in fermented food and are generally considered safe. LAB regulate soil organic matter and the biochemical cycle, detoxify hazardous chemicals, and enhance plant health. They are found in decomposing plants, traditional fermented milk products, and normal human gastrointestinal and vaginal flora. Exploring LAB identified in unknown niches may lead to isolating unique species. However, their classification is quite complex, and they are adapted to high sugar concentrations and acidic environments. LAB strains are considered promising candidates for sustainable agriculture, and they promote soil health and fertility. Therefore, they have received much attention regarding sustainable agriculture. LAB metabolites promote plant growth and stimulate shoot and root growth. As fertilizers, LAB can promote biodegradation, accelerate the soil organic content, and produce organic acid and bacteriocin metabolites. However, LAB show an antagonistic effect against phytopathogens, inhibiting fungal and bacterial populations in the rhizosphere and phyllosphere. Several studies have proposed the LAB bioremediation efficiency and detoxification of heavy metals and mycotoxins. However, LAB genetic manipulation and metabolic engineered tools provide efficient cell factories tailor-made to produce beneficial industrial and agro-products. This review discusses lactic acid bacteria advantages and limitations in sustainable agricultural development.

Production of Carminic Acid by Metabolically Engineered Escherichia coli.

Carminic acid is an aromatic polyketide found in scale insects (i.e., Dactylopius coccus) and is a widely used natural red colorant. It has long been produced by the cumbersome farming of insects followed by multistep purification processes. Thus, there has been much interest in producing carminic acid by the fermentation of engineered bacteria. Here we report the complete biosynthesis of carminic acid from glucose in engineered Escherichia coli. We first optimized the type II polyketide synthase machinery from Photorhabdus luminescens, enabling a high-level production of flavokermesic acid upon coexpression of the cyclases ZhuI and ZhuJ from Streptomyces sp. R1128. To discover the enzymes responsible for the remaining two reactions (hydroxylation and C-glucosylation), biochemical reaction analyses were performed by testing enzyme candidates reported to perform similar reactions. The two identified enzymes, aklavinone 12-hydroxylase (DnrF) from Streptomyces peucetius and C-glucosyltransferase (GtCGT) from Gentiana triflora, could successfully perform hydroxylation and C-glucosylation of flavokermesic acid, respectively. Then, homology modeling and docking simulations were performed to enhance the activities of these two enzymes, leading to the generation of beneficial mutants with 2-5-fold enhanced conversion efficiencies. In addition, the GtCGT mutant was found to be a generally applicable C-glucosyltransferase in E. coli, as was showcased by the successful production of aloesin found in Aloe vera. Simple metabolic engineering followed by fed-batch fermentation resulted in 0.63 ± 0.02 mg/L of carminic acid production from glucose. The strategies described here will be useful for the design and construction of biosynthetic pathways involving unknown enzymes and consequently the production of diverse industrially important natural products.

Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria.

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only in Escherichia coli and Saccharomyces cerevisiae and require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria: E. coli, Pseudomonas putida, and Corynebacterium glutamicum RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor in E. coli Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level in E. coli These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple different E. coli strains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA-derived chemicals.

Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex gene expression knockdown.

Synthetic small regulatory RNA (sRNA) can efficiently downregulate target gene expression at translational level in metabolic engineering, but cannot be used in engineered strain already having incompatible plasmid(s). To address this problem and make the sRNA gene expression modulation platform universally applicable, we report the development and applications of expanded synthetic sRNA expression platforms for rapid, multiplexed and genome-scale target gene knockdown in engineered Escherichia coli. As proof-of-concept, high performance strains capable of producing L-proline (54.1 g l-1) and L-threonine (22.9 g l-1) are rapidly developed by combinatorial knockdown of up to three genes via one-step co-transformation of sRNA expression vectors. Furthermore, a genome-scale sRNA library targeting 1,858 E. coli genes is employed to construct crude violacein (5.19 g l-1) and indigo (135 mg l-1) producers by high-throughput colorimetric screening. These examples demonstrate that the expanded sRNA expression vectors developed here enables rapid development of chemical overproducers regardless of plasmid compatibility.

CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum.

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum.

Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs.

Synthetic sRNAs allow knockdown of target genes at translational level, but have been restricted to a limited number of bacteria. Here, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. BHR-sRNA is tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria, with >50% of target gene knockdown achieved in 12 bacterial species. For medical applications, virulence factors in Staphylococcus epidermidis and Klebsiella pneumoniae are knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, high performance Corynebacterium glutamicum strains capable of producing valerolactam (bulk chemical) and methyl anthranilate (fine chemical) are developed by combinatorial knockdown of target genes. A genome-scale sRNA library covering 2959 C. glutamicum genes is constructed for high-throughput colorimetric screening of indigoidine (natural colorant) overproducers. The BHR-sRNA platform will expedite engineering of diverse bacteria of both industrial and medical interest.

Metabolic and cellular engineering for the production of natural products.

Increased awareness of the environmental and health concerns of consuming chemically synthesized products has led to a rising demand for natural products that are greener and more sustainable. Despite their importance, however, industrial-scale production of natural products has been challenging due to the low yield and high cost of the bioprocesses. To cope with this problem, systems metabolic engineering has been employed to efficiently produce natural products from renewable biomass. Here, we review the recent systems metabolic engineering strategies employed for enhanced production of value-added natural products, together with accompanying examples. Particular focus is set on systems-level engineering and cell physiology engineering strategies. Future perspectives are also discussed.

Production of Rainbow Colorants by Metabolically Engineered Escherichia coli.

There has been much interest in producing natural colorants to replace synthetic colorants of health concerns. Escherichia coli has been employed to produce natural colorants including carotenoids, indigo, anthocyanins, and violacein. However, production of natural green and navy colorants has not been reported. Many natural products are hydrophobic, which are accumulated inside or on the cell membrane. This causes cell growth limitation and consequently reduces production of target chemicals. Here, integrated membrane engineering strategies are reported for the enhanced production of rainbow colorants-three carotenoids and four violacein derivatives-as representative hydrophobic natural products in E. coli. By integration of systems metabolic engineering, cell morphology engineering, inner- and outer-membrane vesicle formation, and fermentation optimization, production of rainbow colorants are significantly enhanced to 322 mg L-1 of astaxanthin (red), 343 mg L-1 of ß-carotene (orange), 218 mg L-1 of zeaxanthin (yellow), 1.42 g L-1 of proviolacein (green), 0.844 g L-1 of prodeoxyviolacein (blue), 6.19 g L-1 of violacein (navy), and 11.26 g L-1 of deoxyviolacein (purple). The membrane engineering strategies reported here are generally applicable to microbial production of a broader range of hydrophobic natural products, contributing to food, cosmetic, chemical, and pharmaceutical industries.

Escherichia coli as a platform microbial host for systems metabolic engineering.

Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

Metabolic Engineering of Escherichia coli for Natural Product Biosynthesis.

Natural products are widely employed in our daily lives as food additives, pharmaceuticals, nutraceuticals, and cosmetic ingredients, among others. However, their supply has often been limited because of low-yield extraction from natural resources such as plants. To overcome this problem, metabolically engineered Escherichia coli has emerged as a cell factory for natural product biosynthesis because of many advantages including the availability of well-established tools and strategies for metabolic engineering and high cell density culture, in addition to its high growth rate. We review state-of-the-art metabolic engineering strategies for enhanced production of natural products in E. coli, together with representative examples. Future challenges and prospects of natural product biosynthesis by engineered E. coli are also discussed.

Broad-Spectrum Gene Repression Using Scaffold Engineering of Synthetic sRNAs.

Gene expression regulation in broad-spectrum range is critical for constructing cell factories and genetic circuits to balance and control system-wide fluxes. Synthetic small regulatory RNAs (sRNAs) effectively regulate gene expression at the translational level by modulating an mRNA-binding chance and sRNA abundance; however, it can control target gene expression only within the limit of the intrinsic repression ability of sRNAs. Here, we systematically mutated a SgrS scaffold as a model sRNA by dividing the Hfq-binding module of the sRNA into the three regions: the A/U-rich sequence, the stem, and the hairpin loop, and examined how efficiently the mutants suppressed DsRed2 expression. By doing this, we found that a scaffold with an altered A/U-rich sequence (CUUU) and stem length and that with altered A/U-rich sequence (GCAC) showed a 3-fold stronger and a 3-fold weaker repression than the original scaffold, respectively. For practical application of altered scaffolds, proof-of-concept experiments were performed by constructing a library of 67 synthetic sRNAs with the strongest scaffold, each one targeting a different rationally selected gene, and using this library to enhance cadaverine production in Escherichia coli, yielding in 27% increase (1.67 g/L in flask cultivation, 13.7 g/L in fed-batch cultivation). Synthetic sRNAs with engineered sRNA scaffolds could be useful in modulating gene expression for strain improvement.

Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering.

Metabolic engineering allows development of microbial strains efficiently producing chemicals and materials, but it requires much time, effort, and cost to make the strains industrially competitive. Systems metabolic engineering, which integrates tools and strategies of systems biology, synthetic biology, and evolutionary engineering with traditional metabolic engineering, has recently been used to facilitate development of high-performance strains. The past decade has witnessed this interdisciplinary strategy continuously being improved toward the development of industrially competitive overproducer strains. In this article, current trends in systems metabolic engineering including tools and strategies are reviewed, focusing on recent developments in selection of host strains, metabolic pathway reconstruction, tolerance enhancement, and metabolic flux optimization. Also, future challenges and prospects are discussed.

Metabolic engineering of Escherichia coli for the production of indirubin from glucose.

Indirubin is an indole alkaloid that can be used to treat various diseases including granulocytic leukemia, cancer, and Alzheimer's disease. Microbial production of indirubin has so far been achieved by supplementation of rather expensive substrates such as indole or tryptophan. Here, we report the development of metabolically engineered Escherichia coli strain capable of producing indirubin directly from glucose. First, the Methylophaga aminisulfidivorans flavin-containing monooxygenase (FMO) and E. coli tryptophanase (TnaA) were introduced into E. coli in order to complete the biosynthetic pathway from tryptophan to indirubin. Further engineering was performed through rational strategies including disruption of the regulatory repressor gene trpR and removal of feedback inhibitions on AroG and TrpE. Then, combinatorial approach was employed by systematically screening eight genes involved in the common aromatic amino acid pathway. Moreover, availability of the aromatic precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate, was enhanced by inactivating the pykF (pyruvate kinase I) and pykA (pyruvate kinase II) genes, and by overexpressing the tktA gene (encoding transketolase), respectively. Fed-batch fermentation of the final engineered strain led to production of 0.056 g/L of indirubin directly from glucose. The metabolic engineering and synthetic biology strategies reported here thus allows microbial fermentative production of indirubin from glucose.

Systems metabolic engineering as an enabling technology in accomplishing sustainable development goals.

With pressing issues arising in recent years, the United Nations proposed 17 Sustainable Development Goals (SDGs) as an agenda urging international cooperations for sustainable development. In this perspective, we examine the roles of systems metabolic engineering (SysME) and its contribution to improving the quality of life and protecting our environment, presenting how this field of study offers resolutions to the SDGs with relevant examples. We conclude with offering our opinion on the current state of SysME and the direction it should move forward in the generations to come, explicitly focusing on addressing the SDGs.

Systems Metabolic Engineering of Escherichia coli.

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

Systems Metabolic Engineering of Escherichia coli.

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.