Metabolic engineering study on biosynthesis of 4-hydroxybenzyl alcohol from L-tyrosine in Escherichia coli / 中国中药杂志

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.

Application and population control strategy of microbial modular co-culture engineering / 生物工程学报

Traditional methods of microbial synthesis usually rely on a single engineered strain to synthesize the target product through metabolic engineering. The key cofactors, precursors and energy are produced by the introduced complex synthetic pathways. This would increase the physiological burden of engineering strains, resulting in a decrease in the yield of target products. The modular co-culture engineering has become an attractive solution for effective heterologous biosynthesis, where product yield can be greatly improved. In the modular co-culture engineering, the coordination between the population of different modules is essential for increasing the production efficiency. This article summarized recent advances in the application of modular co-culture engineering and population control strategies.

Recent progress in ergothioneine biosynthesis: a review / 生物工程学报

Ergothioneine is a multifunctional physiological cytoprotector, with broad application in foods, beverage, medicine, cosmetics and so on. Biosynthesis is an increasingly favored method in the production of ergothioneine. This paper summarizes the new progress in the identification of key pathways, the mining of key enzymes, and the development of natural edible mushroom species and high-yield engineering strains for ergothioneine biosynthesis in recent years. Through this review, we aim to reveal the molecular mechanism of ergothioneine biosynthesis and then employ the methods of fermentation engineering, metabolic engineering, and synthetic biology to greatly increase the yield of ergothioneine.

Graph-based and constraint-based heterologous metabolic pathway design methods and application / 生物工程学报

It is among the goals in metabolic engineering to construct microbial cell factories producing high-yield and high value-added target products, and an important solution is to design efficient synthetic pathway for the target products. However, due to the difference in metabolic capacity among microbial chassises, the available substrate and the yielded products are limited. Therefore, it is urgent to design related metabolic pathways to improve the production capacity. Existing metabolic engineering approaches to designing heterologous pathways are mainly based on biological experience, which are inefficient. Moreover, the yielded results are in no way comprehensive. However, systems biology provides new methods for heterologous pathway design, particularly the graph-based and constraint-based methods. Based on the databases containing rich metabolism information, they search for and uncover possible metabolic pathways with designated strategy (graph-based method) or algorithm (constraint-based method) and then screen out the optimal pathway to guide the modification of strains. In this paper, we reviewed the databases and algorithms for pathway design, and the applications in metabolic engineering and discussed the strengths and weaknesses of existing algorithms in practical application, hoping to provide a reference for the selection of optimal methods for the design of product synthesis pathway.

Tolerance engineering regulates stress resistance of microbial cell factory / 生物工程学报

The production efficiency of microbial cell factory is determined by the growth performance, product synthetic capacity, and stress resistance of the strain. Strengthening the stress resistance is the key point to improve the production efficiency of microbial cell factory. Tolerance engineering is based on the response mechanism of microbial cell factory to resist stress. Specifically, it consolidates the cell wall-cell membrane barrier to enhance the defense against stress, accelerates the stress response to improve the damage repair, and creates tolerance evolutionary tools to screen industrial microorganisms with enhanced robustness. We summarize the regulation strategies and forecast the prospects of tolerance engineering, which plays an important role in the microbial cell factories for sustainable production of natural products and bulk chemicals.

Production of carboxylic acids by metabolically engineered Yarrowia lipolytica: a review / 生物工程学报

Yarrowia lipolytica is a non-conventional yeast with unique physiological and metabolic characteristics. It is suitable for production of various products due to its natural ability to utilize a variety of inexpensive carbon sources, excellent tolerance to low pH, and strong ability to secrete metabolites. Currently, Y. lipolytica has been demonstrated to produce a wide range of carboxylic acids with high efficiency. This article summarized the progress in engineering Y. lipolytica to produce various carboxylic acids by using metabolic engineering and synthetic biology approaches. The current bottlenecks and solutions for high-level production of carboxylic acids by engineered Y. lipolytica were also discussed, with the aim to provide useful information for relevant studies in this field.

Construction and application of microbial cell factories for unnatural amino acids / 生物工程学报

Unnatural amino acids are widely used in medicine, pesticide, material, and other industries and the green and efficient synthesis has attracted a lot of attention. In recent years, with the rapid development of synthetic biology, microbial cell factories have become a promising means for biosynthesis of unnatural amino acids. This study reviewed the construction and application of microbial cell factories for unnatural amino acid, including the synthetic pathway reconstruction, design/modification of key enzymes and their coordinated regulation with precursors, blocking of competitive alternative pathways, and construction of cofactor circulation systems. Meanwhile, on the basis of the new principles for designing the microbial cell factories, new biosynthetic pathways adapted to cells and the production environment, as well as new biomanufacturing system established based on cell adaptive evolution and intelligent fermentation regulation, we looked forward to the further construction and application of microbial cell factories for industrial bio-production.

Biomanufacturing driven by engineered microbes / 生物工程学报

This article summarized the reviews and research articles published in Chinese Journal of Biotechnology in the field of biomanufacturing in 2021. The article covered major chassis cells such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, filamentous fungi, non-model bacteria and non-conventional yeasts. Moreover, this article summarized the advances in the production of amino acids, organic acids, vitamins, higher alcohols, natural compounds (terpenoids, flavonoids, alkaloids), antibiotics, enzymes and enzyme-catalyzed products, biopolymers, as well as the utilization of biomass and one-carbon materials. The key technologies used in the construction of cell factories, such as regulation, evolution, and high-throughput screening, were also included. This article may help the readers better understand the R & D trend in biomanufacturing driven by engineered microbes.

Construction and application of a synthetic promoter library for Corynebacterium glutamicum / 生物工程学报

Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter Ptrc. Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif "TANNNT" to the 3' end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter PodhA. The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.

Construction and optimization of ergothioneine-producing Escherichia coli / 生物工程学报

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serAT410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serAT410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.

Production of fatty acids by engineered Ogataea polymorpha / 生物工程学报

Fatty acids (FA) are widely used as feed stocks for the production of cosmetics, personal hygiene products, lubricants and biofuels. Ogataea polymorpha is considered as an ideal chassis for bio-manufacturing, due to its outstanding characteristics such as methylotroph, thermal-tolerance and wide substrate spectrum. In this study, we harnessed O. polymorpha for overproduction of fatty acids by engineering its fatty acid metabolism and optimizing the fermentation process. The engineered strain produced 1.86 g/L FAs under the optimized shake-flask conditions (37℃, pH 6.4, a C/N ratio of 120 and an OD600 of seed culture of 6-8). The fed-batch fermentation process was further optimized by using a dissolved oxygen (DO) control strategy. The C/N ratio of initial medium was 17.5, and the glucose medium with a C/N ratio of 120 was fed when the DO was higher than 30%. This operation resulted in a titer of 18.0 g/L FA, indicating the potential of using O. polymorpha as an efficient cell factory for the production of FA.

A CRISPR/dCpf1-based transcriptional repression system for Gluconobacter oxydans / 生物工程学报

Gluconobacter oxydans are widely used in industrial due to its ability of oxidizing carbohydrate rapidly. However, the limited gene manipulation methods and less of efficient gene editing tools impose restrictions on its application in industrial production. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been widely used in genome editing and transcriptional regulation which improves the efficiency of genome editing greatly. Here we constructed a CRISPR/dCpf1-mediated gene transcriptional repression system, the expression of a nuclease inactivation Cpf1 protein (dCpf1) in Gluconobacter oxydans together with a 19 nt direct repeats showed effective repression in gene transcription. This system in single gene repression had strong effect and the relative repression level had been increased to 97.9%. While it could be applied in multiplex gene repression which showed strong repression ability at the same time. Furthermore, this system was used in the metabolic pathway of L-sorbose and the regulatory of respiratory chain. The development of CRISPR transcriptional repression system effectively covered the shortage of current gene regulation methods in G. oxydans and provided an efficient gene manipulation tool for metabolic engineering modification in G. oxydans.

Engineering Saccharomyces cerevisiae for efficient production of glucaric acid / 生物工程学报

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.

Engineering the glycogen metabolism in cyanobacterial photosynthetic cell factories: a review / 生物工程学报

Cyanobacteria are important photosynthetic autotrophic microorganisms and are considered as one of the most promising microbial chassises for photosynthetic cell factories. Glycogen is the most important natural carbon sink of cyanobacteria, playing important roles in regulating its intracellular carbon distributions. In order to optimize the performances of cyanobacterial photosynthetic cell factories and drive more photosynthetic carbon flow toward the synthesis of desired metabolites, many strategies and approaches have been developed to manipulate the glycogen metabolism in cyanobacteria. However, the disturbances on glycogen metabolism usually cause complex effects on the physiology and metabolism of cyanobacterial cells. Moreover, the effects on synthesis efficiencies of different photosynthetic cell factories usually differ. In this manuscript, we summarized the recent progress on engineering cyanobacterial glycogen metabolism, analyzed and compared the physiological and metabolism effects caused by engineering glycogen metabolism in different cyanobacteria species, and prospected the future trends of this strategy on optimizing cyanobacterial photosynthetic cell factories.

Development of metabolic models with multiple constraints: a review / 生物工程学报

Constraint-based genome-scale metabolic network models (genome-scale metabolic models, GEMs) have been widely used to predict metabolic phenotypes. In addition to stoichiometric constraints, other constraints such as enzyme availability and thermodynamic feasibility may also limit the cellular phenotype solution space. Recently, extended GEM models considering either enzymatic or thermodynamic constraints have been developed to improve model prediction accuracy. This review summarizes the recent progresses on metabolic models with multiple constraints (MCGEMs). We presented the construction methods and various applications of MCGEMs including the simulation of gene knockout, prediction of biologically feasible pathways and identification of bottleneck steps. By integrating multiple constraints in a consistent modeling framework, MCGEMs can predict the metabolic bottlenecks and key controlling and modification targets for pathway optimization more precisely, and thus may provide more reliable design results to guide metabolic engineering of industrially important microorganisms.

The recent advances in developing gene editing and expression tools and the synthesis of natural products in Yarrowia lipolytica / 生物工程学报

Yarrowia lipolytica, as an important oleaginous yeast, has been widely used in metabolic engineering. Y. lipolytica is considered as an ideal host for the production of natural products such as terpenes, polyketides and flavonoids, due to its ability to utilize a variety of hydrophobic substrates, high stress tolerance to acid and salt, high flux of tricarboxylic acid cycle and the ability in providing abundant the common precursor acetyl-CoA. Recently, more and more tools for genetic editing, gene expression and regulation has been developed in Y. lipolytica, which facilitate the metabolic engineering of Y. lipolytica for bio-manufacturing. In this review, we summarized the recent progresses in developing gene expression and natural product synthesis in Y. lipolytica, and also discussed the challenges and possible solutions in heterologous synthesis of natural products in this yeast.

Oxidosqualene cyclases in triterpenoids biosynthesis: a review / 生物工程学报

Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.

Microbial synthesis of monoterpenoids: a review / 生物工程学报

Monoterpenoids that belong to the terpenoids family are usually volatile and have strong aroma. Some monoterpenoids also have antioxidant, antibacterial and anti-inflammatory activities, which make them important raw materials for medicine, food and cosmetics industry. In recent years, the heterologous synthesis of monoterpenoids by microorganisms has attracted extensive attention. However, its large-scale application is greatly hampered by the low yield and high production cost. Nowadays, the rapid development of synthetic biology provides new approaches for enhancing the production of monoterpenoids by microorganisms. Different kinds of recombinant strains can be obtained via engineering of microbial cells to produce a variety of monoterpenoids with different properties. This paper summarized the latest strategies and progress in the application of synthetic biology to produce monoterpenoids by microorganisms, including the design and modification of biosynthetic pathway, as well as the design and optimization of high-yield monoterpenoids producing chassis cells.

Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid / 生物工程学报

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

Heterogeneous expression of DOPA decarboxylase to improve the production of dopamine in Escherichia coli / 生物工程学报

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.