Metabolic Engineering Strategies Research Articles

Increased Flux of Lipid Metabolism Enhances Bioethanol Fermentability and Inhibitor Tolerance of Xylose-Utilizing Zymomonas mobilis

The microbial production of fuel ethanol is an attractive and sustainable biotechnological approach. This study presents a metabolic engineering strategy of Zymomonas mobilis aimed at coproducing bioethanol and fatty acids. The increased flux of fatty acids stabilizes the cell membrane and thus counteracts the progressively higher ethanol toxicity. In a glucose medium, the highest ethanol titer achieved was 146.7 g/kg of broth, surpassing the wild-type Z. mobilis CP4 and angel yeast by 30% and 45%, respectively. The recombinant strain exhibited a total fatty acid titer of 0.4 g/L from 230 g/L total sugar solution (5 L bioreactor), representing a 12-fold increase compared to the wild-type Z. mobilis CP4. Furthermore, when using a 4:2:1 mixture of glucose: xylose: mannose (w/v), an ethanol concentration of 142.8 g/kg of broth was attained, only 2.66% lower than that of the glucose-only medium. These findings highlight the enormous potential of this genetically engineered strain for the sustainable production of ethanol and fatty acids from lignocellulosic renewable carbon sources.

Engineering Escherichia coli BL21 (DE3) for high-yield production of germacrene A, a precursor of β-elemene via combinatorial metabolic engineering strategies.

β-elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant-derived natural chemical, biologically engineering microorganisms to producegermacrene A to be converted to β-elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to β-elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high-efficient β-elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl-coA, pyruvate, and glyceraldehyde-3-phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NSY305N was obtained via error-prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of β-elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of β-elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4-L fed-batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl-coA-derived and pyruvate-derived molecules cost-effectively.

Multivariate modular metabolic engineering for enhanced l-methionine biosynthesis in Escherichia coli

Backgroundl-Methionine is the only bulk amino acid that has not been industrially produced by the fermentation method. Due to highly complex and strictly regulated biosynthesis, the development of microbial strains for high-level l-methionine production has remained challenging in recent years.ResultsBy strengthening the l-methionine terminal synthetic module via site-directed mutation of l-homoserine O-succinyltransferase (MetA) and overexpression of metAfbr, metC, and yjeH, l-methionine production was increased to 1.93 g/L in shake flask fermentation. Deletion of the pykA and pykF genes further improved l-methionine production to 2.51 g/L in shake flask fermentation. Computer simulation and auxotrophic experiments verified that during the synthesis of l-methionine, equimolar amounts of l-isoleucine were accumulated via the elimination reaction of cystathionine γ-synthetase MetB due to the insufficient supply of l-cysteine. To increase the supply of l-cysteine, the l-cysteine synthetic module was strengthened by overexpression of cysEfbr, serAfbr, and cysDN, which further increased the production of l-methionine by 52.9% and significantly reduced the accumulation of the byproduct l-isoleucine by 29.1%. After optimizing the addition of ammonium thiosulfate, the final metabolically engineered strain MET17 produced 21.28 g/L l-methionine in 64 h with glucose as the carbon source in a 5 L fermenter, representing the highest l-methionine titer reported to date.ConclusionsIn this study, a high-efficiency strain for l-methionine production was derived from wild-type Escherichia coli W3110 by rational metabolic engineering strategies, providing an efficient platform for the industrial production of l-methionine.

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.

Manipulation of IME4 expression, a global regulation strategy for metabolic engineering in Saccharomyces cerevisiae.

Metabolic engineering has been widely used for production of natural medicinal molecules. However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N6-Methyladenosine (m6A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m6A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4 (the yeast m6A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner. Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m6A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.

Microbial Production of Mevalonate.

Mevalonate, an important intermediate product of the mevalonate pathway, has a broad spectrum of applications. With the rapid growth of metabolic engineering and synthetic biology, mevalonate biosynthesis by microorganisms is feasible and holds great promise in the future. In this review, we summarize the applications of mevalonate and its derivatives and describe the biosynthesis pathways of mevalonate. The current status of mevalonate biosynthesis is also detailed with an emphasis on metabolic engineering strategies to enhance mevalonate production in typical industrial organisms, including Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida, suggesting new insights for the efficient production of biosynthesized mevalonate.

Efficient production of natural sunscreens shinorine, porphyra-334, and mycosporine-2-glycine in Saccharomyces cerevisiae

Mycosporine-like amino acids (MAAs) are promising natural sunscreens mainly produced in marine organisms. Until now, metabolic engineering efforts to produce MAAs in heterologous hosts have mainly focused on shinorine production, and the low production levels are still not suitable for industrial applications. In this study, we successfully developed Saccharomyces cerevisiae strains that can efficiently produce various disubstituted MAAs, including shinorine, porphyra-334, and mycosporine-2-glycine (M2G), which are formed by conjugating serine, threonine, and glycine to mycosporine-glycine (MG), respectively. We first generated an MG-producing strain by multiple integration of the biosynthetic genes from cyanobacteria and applying metabolic engineering strategies to increase sedoheptulose-7-phosphate pool, a substrate for MG production. Next, five mysD genes from cyanobacteria, which encode D-Ala-D-Ala ligase homologues that conjugate an amino acid to MG, were introduced into the MG-producing strain to determine the substrate preference of each MysD enzyme. MysDs from Lyngbya sp., Nostoclinckia, and Euhalothece sp. showed high specificity toward serine, threonine, and glycine, resulting in efficient production of shinorine, porphyra-334, and M2G, respectively. This is the first report on the production of porphyra-334 and M2G in S. cerevisiae. Furthermore, we identified that the substrate specificity of MysD was determined by the omega loop region of 43–45 amino acids predicted based on its structural homology to a D-Ala-D-Ala ligase from Thermus thermophilus involved in peptidoglycan biosynthesis. The substrate specificities of two MysD enzymes were interchangeable by swapping the omega loop region. Using the engineered strain expressing mysD from Lyngbya sp. or N. linckia, up to 1.53 g/L shinorine or 1.21 g/L porphyra-334 was produced by fed-batch fermentation in a 5-L bioreactor, the highest titer reported so far. These results suggest that S. cerevisiae is a promising host for industrial production of different types of MAAs, providing a sustainable and eco-friendly alternative for the development of natural sunscreens.

Metabolic engineering of Escherichia coli for the efficient production of 5-hydroxyvaleric acid

There is increasing industrial demand for five-carbon platform chemicals, particularly 5-hydroxyvaleric acid (5-HV), a widely used building block for the synthesis of polyesters and polyurethanes. Here, we developed an efficient 5-HV microbial producer by systems metabolic engineering of Escherichia coli W3110. To derive 5-HV, Pseudomonas putidal-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA), E. coli 4-aminobutyrate aminotransferase (gabT), and different aldehyde reductase genes were introduced to E. coli W3110 to convert l-lysine hydrochloride to the target product. Three aldehyde reductases, including YajO from P. putida and YahK and YqhD from E. coli, were examined, revealing that the overexpression of aldehyde reductases was critical for 5-HV production and YajO was first reported for efficient production of 5-HV. The E. coli 5-aminovalerate exporter encoded by gabP and E. coli lysine-specific permease encoded by lysP were expressed to further enhance 5-HV production. In addition, a hok/sok toxin/antitoxin system based on the E. coli XL1-Blue genome was inserted to the dual-vector system to decrease plasmid loss. Finally, 5.59 g/L of 5-HV with a yield of 0.96 mol/mol (5-HV/l-lysine hydrochloride) by shake flask cultivation of the engineered E. coli W3110 and 35.6 g/L of 5-HV with a yield of 0.92 mol/mol (5-HV/l-lysine hydrochloride) by fed fermentation were achieved respectively. The developed metabolic engineering strategies provide a basis for the efficient and sustainable bio-based production of 5-HV.

Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast.

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

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.

Efficient Synthesis of Limonene in Saccharomyces cerevisiae Using Combinatorial Metabolic Engineering Strategies

Limonene is a volatile monoterpene compound that is widely used in food additives, pharmaceutical products, fragrances, and toiletries. We herein attempted to perform efficient biosynthesis of limonene in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, we conducted de novo synthesis of limonene in S. cerevisiae and achieved a titer of 46.96 mg/L. Next, by dynamic inhibition of the competitive bypass of key metabolic branches regulated by ERG20 and optimization of the copy number of tLimS, a greater proportion of the metabolic flow was directed toward limonene synthesis, achieving a titer of 640.87 mg/L. Subsequently, we enhanced the acetyl-CoA and NADPH supply, which increased the limonene titer to 1097.43 mg/L. Then, we reconstructed the limonene synthesis pathway in the mitochondria. Dual regulation of cytoplasmic and mitochondrial metabolism further increased the limonene titer to 1586 mg/L. After optimization of the process of fed-batch fermentation, the limonene titer reached 2.63 g/L, the highest ever reported in S. cerevisiae.

Comparative genomic analysis of Methylocystis sp. MJC1 as a platform strain for polyhydroxybutyrate biosynthesis.

Biodegradable polyhydroxybutyrate (PHB) can be produced from methane by some type II methanotroph such as the genus Methylocystis. This study presents the comparative genomic analysis of a newly isolated methanotroph, Methylocystis sp. MJC1 as a biodegradable PHB-producing platform strain. Methylocystis sp. MJC1 accumulates up to 44.5% of PHB based on dry cell weight under nitrogen-limiting conditions. To facilitate its development as a PHB-producing platform strain, the complete genome sequence of Methylocystis sp. MJC1 was assembled, functionally annotated, and compared with genomes of other Methylocystis species. Phylogenetic analysis has shown that Methylocystis parvus to be the closest species to Methylocystis sp. MJC1. Genome functional annotation revealed that Methylocystis sp. MJC1 contains all major type II methanotroph biochemical pathways such as the serine cycle, EMC pathway, and Krebs cycle. Interestingly, Methylocystis sp. MJC1 has both particulate and soluble methane monooxygenases, which are not commonly found among Methylocystis species. In addition, this species also possesses most of the RuMP pathway reactions, a characteristic of type I methanotrophs, and all PHB biosynthetic genes. These comparative analysis would open the possibility of future practical applications such as the development of organism-specific genome-scale models and application of metabolic engineering strategies to Methylocystis sp. MJC1.

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation.

Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72°C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85°C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95°C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75°C and 95°C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol. IMPORTANCE Previously, the highest temperature for biological ethanol production was 85°C. Here, we have engineered ethanol production at 95°C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75°C and 95°C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.

Towards microalga-based superfoods: heterologous expression of zeolin in Chlamydomonas reinhardtii.

Microalgae are unicellular photosynthetic organisms that can be grown in artificial systems to capture CO2, release oxygen, use nitrogen- and phosphorus-rich wastes, and produce biomass and bioproducts of interest including edible biomass for space exploration. In the present study, we report a metabolic engineering strategy for the green alga Chlamydomonas reinhardtii to produce high-value proteins for nutritional purposes. Chlamydomonas reinhardtii is a species approved by the U.S. Food and Drug Administration (FDA) for human consumption, and its consumption has been reported to improve gastrointestinal health in both murine models and humans. By utilizing the biotechnological tools available for this green alga, we introduced a synthetic gene encoding a chimeric protein, zeolin, obtained by merging the γ-zein and phaseolin proteins, in the algal genome. Zein and phaseolin are major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris) that accumulate in the endoplasmic reticulum (ER) and storage vacuoles, respectively. Seed storage proteins have unbalanced amino acid content, and for this reason, need to be complemented with each other in the diet. The chimeric recombinant zeolin protein represents an amino acid storage strategy with a balanced amino acid profile. Zeolin protein was thus efficiently expressed in Chlamydomonas reinhardtii; thus, we obtained strains that accumulate this recombinant protein in the endoplasmic reticulum, reaching a concentration up to 5.5 fg cell-1, or secrete it in the growth medium, with a titer value up to 82 µg/L, enabling the production of microalga-based super-food.

Applications of the Whole-Cell System in the Efficient Biosynthesis of Heme.

Heme has a variety of functions, from electronic reactions to binding gases, which makes it useful in medical treatments, dietary supplements, and food processing. In recent years, whole-cell system-based heme biosynthesis methods have been continuously explored and optimized as an alternative to the low-yield, lasting, and adverse ecological environment of chemical synthesis methods. This method relies on two biosynthetic pathways of microbial precursor 5-aminolevulinic acid (C4, C5) and three known downstream biosynthetic pathways of heme. This paper reviews the genetic and metabolic engineering strategies for heme production in recent years by optimizing culture conditions and techniques from different microorganisms. Specifically, we summarized and analyzed the possibility of using biosensors to explore new strategies for the biosynthesis of heme from the perspective of synthetic biology, providing a new direction for future exploration.

De novo biosynthesis of sakuranetin from glucose by engineered Saccharomyces cerevisiae.

Sakuranetin is a plant-natural product, which has increasingly been utilized in cosmetic and pharmaceutical industries for its extensive anti-inflammatory, anti-tumor, and immunomodulatory effects. Sakuranetin was mostly produced by extraction technology from plants, which is limited to natural conditions and biomass supply. In this study, a de novo biosynthesis pathway of sakuranetin by engineered S. cerevisiae was constructed. After a series of heterogenous gene integration, a biosynthetic pathway of sakuranetin from glucose was successfully constructed in S. cerevisiae whose sakuranetin yield reached only 4.28 mg/L. Then, a multi-module metabolic engineering strategy was applied for improving sakuranetin yield in S. cerevisiae: (1) adjusting the copy number of sakuranetin synthesis genes, (2) removing the rate-limiting factor of aromatic amino acid pathway and optimizing the synthetic pathway of aromatic amino acids to enhance the supply of carbon flux for sakuranetin, and (3) introducing acetyl-CoA carboxylase mutants ACC1S659A,S1157A and knocking out YPL062W to strengthen the supply of malonyl-CoA which is another synthetic precursor of sakuranetin. The resultant mutant S. cerevisiae exhibited a more than tenfold increase of sakuranetin titer (50.62 mg/L) in shaking flasks. Furthermore, the sakuranetin titer increased to 158.65 mg/L in a 1-L bioreactor. To our knowledge, it is the first report on the sakuranetin de novo synthesis from glucose in S. cerevisiae. KEY POINTS: • De novo biosynthesis of sakuranetin was constructed by engineered S. cerevisiae. • Sakuranetin production was enhanced by multi-module metabolic engineering strategy. • It is the first report on the sakuranetin de novo synthesis in S. cerevisiae.

Combining in silico and in vitro approaches to understand the involvement of MEP and shikimate pathway in Agrobacterium tumefaciens for enhanced CoQ10 production.

To carry out integrated comparative metabolic pathway analysis to understand Coenzyme Q10 (CoQ10) production in Agrobacterium tumefaciens. Comparative analysis of the CoQ10 metabolic pathways in 10 organisms using a G2KO program and KEGG database shed light on the completeness of the production pathway in A. tumefaciens. The specific role of the key precursors and the enzymes in the metabolic network was further validated using pathway inhibitors and enhancers. While the use of fosmidomycin and glyphosate displayed 54.54%-99% suppression in CoQ10 production, supplementation of polyprenyl pyrophosphate from the MEP (methylerythritol 4-phosphate) pathway and 4-hydroxybenzoate precursor from the shikimate pathway, enhanced CoQ10 production by 2.3-fold. The present study provides a comprehensive understanding of the CoQ10 biosynthetic pathway in A. tumefaciens, which would assist rational metabolic engineering strategies for boosting CoQ10 biosynthesis.

The current situations and limitations of genetic engineering in cyanobacteria: a mini review

Cyanobacteria are an ancient group of photoautotrophic prokaryotes, and play an essential role in the global carbon cycle. They are also model organisms for studying photosynthesis and circadian regulation, and metabolic engineering and synthetic biology strategies grants light-driven biotechnological applications to cyanobacteria, especially for engineering cyanobacteria cells to achieve an efficient light-driven system for synthesizing any product of interest from renewable feedstocks. However, lower yield limits the potential of industrial application of cyanobacterial synthetic biology, and some key limitations must be overcome to realize the full biotechnological potential of these versatile microorganisms. Although genetic engineering toolkits for cyanobacteria have made some progress, the tools available still lag behind conventional heterotrophic microorganism. Consequently, this study describes the current situations and limitations of genetic engineering in cyanobacteria, and further improvements are proposed to improve the output of targeted products. We believe that cyanobacteria-mediated light-driven platforms towards efficient synthesis of green chemicals could unlock a bright future by developing the tools for strain manipulation and novel chassis organisms with excellent performance for biotechnological applications, which could also accelerate the advancement of bio-manufacturing industries.

Microbial Synthesis of Heme b: Biosynthetic Pathways, Current Strategies, Detection, and Future Prospects.

Heme b, which is characterized by a ferrous ion and a porphyrin macrocycle, acts as a prosthetic group for many enzymes and contributes to various physiological processes. Consequently, it has wide applications in medicine, food, chemical production, and other burgeoning fields. Due to the shortcomings of chemical syntheses and bio-extraction techniques, alternative biotechnological methods have drawn increasing attention. In this review, we provide the first systematic summary of the progress in the microbial synthesis of heme b. Three different pathways are described in detail, and the metabolic engineering strategies for the biosynthesis of heme b via the protoporphyrin-dependent and coproporphyrin-dependent pathways are highlighted. The UV spectrophotometric detection of heme b is gradually being replaced by newly developed detection methods, such as HPLC and biosensors, and for the first time, this review summarizes the methods used in recent years. Finally, we discuss the future prospects, with an emphasis on the potential strategies for improving the biosynthesis of heme b and understanding the regulatory mechanisms for building efficient microbial cell factories.

Evaluation of Various Escherichia coli Strains for Enhanced Lycopene Production.

Lycopene is a carotenoid widely used as a food and feed supplement due to its antioxidant, anti-inflammatory, and anti-cancer functions. Various metabolic engineering strategies have been implemented for high lycopene production in Escherichia coli, and for this purpose it was essential to select and develop an E. coli strain with the highest potency. In this study, we evaluated 16 E. coli strains to determine the best lycopene production host by introducing a lycopene biosynthetic pathway (crtE, crtB, and crtI genes cloned from Deinococcus wulumuqiensis R12 and dxs, dxr, ispA, and idi genes cloned from E. coli). The 16 lycopene strain titers diverged from 0 to 0.141 g/l, with MG1655 demonstrating the highest titer (0.141 g/l), while the SURE and W strains expressed the lowest (0 g/l) in an LB medium. When a 2 × YTg medium replaced the MG1655 culture medium, the titer further escalated to 1.595 g/l. These results substantiate that strain selection is vital in metabolic engineering, and further, that MG1655 is a potent host for producing lycopene and other carotenoids with the same lycopene biosynthetic pathway.