Characterization of highly active tyrosine ammonia lyase and its application in biosynthesis of p-coumaric acid / 生物工程学报

p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 ℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.

Advances in the microbial synthesis of aromatic fragrance molecules / 生物工程学报

Aromatic compounds make up a large part of fragrances and are traditionally produced by chemical synthesis and direct extraction from plants. Chemical synthesis depends on petroleum resources and has disadvantages such as causing environment pollutions and harsh reaction conditions. Due to the low content of aromatic compounds in plants and the low yield of direct extraction, plant extractions require large amounts of plant resources that occupy arable land. In recent years, with the development of metabolic engineering and synthetic biology, microbial synthesis of aromatic compounds from renewable resources has become a promising alternative approach to traditional methods. This review describes the research progress on the synthesis of aromatic fragrances by model microorganisms such as Escherichia coli or yeast, including the synthesis of vanillin through shikimic acid pathway and the synthesis of raspberry ketone through polyketide pathway. Moreover, this review highlights the elucidation of native biosynthesis pathways, the construction of synthetic pathways and metabolic regulation for the production of aromatic fragrances by microbial fermentation.

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.

Advances in metabolic engineering for the production of aromatic chemicals / 生物工程学报

Metabolic engineering has been developed for nearly 30 years since the early 1990s, and it has given a great impetus to microbial strain breeding and improvement. Aromatic chemicals are a variety of important chemicals that can be produced by microbial fermentation and are widely used in the pharmaceutical, food, feed, and material industry. Microbial cells can be engineered to accumulate a variety of useful aromatic chemicals in a targeted manner through rational engineering of the biosynthetic pathways of shikimate and the derived aromatic amino acids. This review summarizes the metabolic engineering strategies and biosynthetic pathways for the production of aromatic chemicals developed in the past 30 years, with the aim to provide a valuable reference and promote the research in this field.

Advances in metabolic engineering of non-conventional yeasts / 生物工程学报

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

Preface for special issue on the 30th anniversary of metabolic engineering (2021) / 生物工程学报

Metabolic engineering is the use of recombinant DNA technology, synthetic biology and genome editing to modify the cellular networks including metabolic, gene regulatory, and signaling networks of an organism. It can achieve the desirable goals such as enhanced production of metabolites, and improve the capability of biomanufacturing pharmaceuticals, biofuels and biochemicals as well as other biotechnology products. In order to comprehend the status of metabolic engineering in past 30 years, we published this special issue to review the progress and trends of metabolic engineering from the four aspects of overall development, key technologies, host engineering and product engineering, respectively, for laying the foundation for the further development of metabolic engineering.

Rapid generation of double-layer emulsion droplets based on microfluidic chip / 生物工程学报

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.

Construction of seamless genome editing system for fast-growing Vibrio natriegens / 生物工程学报

Recently, fast-growing Vibrio natriegens, as the great potential chassis, has shown a wide application in synthetic biology. Genome editing is an indispensable tool for genetic modification in synthetic biology. However, genome editing tools with high efficiency and fidelity are still to be developed for V. natriegens synthetic biology. To deal with this problem, the physiological characteristics of 6 V. natriegens strains were evaluated, and CICC 10908 strain with fast and stable growth was selected as the host strain for genome editing study. Then, the natural transformation system of V. natriegens was established and optimized. The efficiencies of optimized natural transformation that integrates antibiotic resistance marker cat-sacB or Kan(R) onto the chromosome of V. natriegens could reach 4×10⁻⁵ and 4×10⁻⁴, respectively. Based on the optimized natural transformation, a double-selection cassette was used to achieve seamless genome editing with high efficiency and fidelity. The positive rates of four different types of genetic manipulation, including gene deletion, complementation, insertion and substitution, were 93.8%, 100%, 95.7% and 100%, respectively. Finally, transformation and elimination of the recombinant plasmid could be easily achieved in V. natriegens. This work provides a seamless genome editing system with high efficiency and fidelity for V. natriegens synthetic biology.

Directed evolution of tyrosine ammonia-lyase to improve the production of p-coumaric acid in Escherichia coli / 生物工程学报

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.

Development and application of a droplet-based microfluidic high-throughput screening of Pichia pastoris / 生物工程学报

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.

Improving the production of 4-aminobenzoic in engineered Escherichia coli by combinatorial regulation / 生物工程学报

Para-aminobenzoate (PABA) is an important chemical for organic synthesis and extensively used in pharmaceutical and dye industry. In recent years, PABA has received increasing attention as a potential component of high-strength polymer. In Escherichia coli, three genes of pabA, pabB and pabC are responsible for PABA production from chorismate in folate synthetic pathway. However, E. coli does not accumulate or accumulates very few amounts of PABA under normal growth condition. In this study, the tyrosine-producing E. coli TYR002 constructed previously was used as the starting strain for developing PABA-producing strain. First, the activity of bifunctional chorismate mutase/prephenate dehydrogenase TyrA in E. coli TYR002 was weakened to reduce the production of tyrosine. Then, three different constitutive promoters were used to regulate the expression of pabA, pabB and pabC in recombinant plasmid which was transformed into E. coli for improving PABA production. The shake-flask fermentation showed that the different combination of constitutive promoters significantly affected the production of PABA, and the highest shake-flask fermentation titer was 0.67 g/L. After further condition optimization, the engineered E. coli produced 6.4 g/L PABA under 5 L fed-batch fermentation. This study could be a good reference for improving microbial production of PABA.

Preface for special issue on industrial biology (2019) / 生物工程学报

Industrial biotechnology promises to make a significant contribution in enabling the sustainable development, and need the solid support from its basic discipline. As the basis of industrial biotechnology, industrial biology is to study the basic laws and mechanisms of biological behavior in industrial environment and to solve the key scientific problems for understanding, designing and constructing the organisms adapted to the application of industrial environment. In order to comprehend the status of industrial biology, we published this special issue to review the progress and trends of industrial biology from the three aspects of industrial protein science, cell science and fermentation science, respectively, for laying the foundation for the development of industrial biotechnology.

Regulation and adaptive evolution of industrial microorganisms towards genetic and environmental disturbances / 生物工程学报

Harnessing industrial microorganisms to utilize renewable feedstocks and meanwhile produce biofuels, bulk chemicals, food ingredients, nutraceuticals, pharmaceuticals, industrial enzymes, etc. is the basis for successful biological industries. Robust traits of industrial microorganisms including high yield and productivity as well as stress tolerance are controlled by sophisticated genetic regulatory networks. Engineering robustness of industrial microorganisms requires systematic and global perturbations at the genome-wide scale to accelerate the accumulation of diversified genotypic mutations, thus generating desirable phenotypes. We review heve the mechanisms of genetic regulation and stress response in robust industrial organisms, the global perturbations and multiplex accelerated evolution at the genome-wide scale, as well as the global perturbation of cellular redox balance. In the future, based on system biology and synthetic biology, more efforts should be further devoted to understanding the mechanisms behind robust traits in industrial microorganisms under industrial niches for modeling and prediction as well as systematic engineering.

Effect of MIG1 and SNF1 deletion on simultaneous utilization of glucose and xylose by Saccharomyces cerevisiae / 生物工程学报

Mig1 and Snf1 are two key regulatory factors involved in glucose repression of Saccharomyces cerevisiae. To enhance simultaneous utilization of glucose and xylose by engineered S. cerevisiae, single and double deletion strains of MIG1 and SNF1 were constructed. Combining shake flask fermentations and transcriptome analysis by RNA-Seq, the mechanism of Mig1 and Snf1 hierarchically regulating differentially expressed genes that might affect simultaneous utilization of glucose and xylose were elucidated. MIG1 deletion did not show any significant effect on co-utilization of mixed sugars. SNF1 deletion facilitated xylose consumption in mixed sugars as well as co-utilization of glucose and xylose, which might be due to that the SNF1 deletion resulted in the de-repression of some genes under nitrogen catabolite repression, thereby favorable to the utilization of nitrogen nutrient. Further deletion of MIG1 gene in the SNF1 deletion strain resulted in the de-repression of more genes under nitrogen catabolite repression and up-regulation of genes involved in carbon central metabolism. Compared with wild type strain, the MIG1 and SNF1 double deletion strain could co-utilize glucose and xylose, and accelerate ethanol accumulation, although this strain consumed glucose faster and xylose slower. Taken together, the MIG1 and SNF1 deletions resulted in up-regulation of genes under nitrogen catabolite repression, which could be beneficial to simultaneous utilization of glucose and xylose. Mig1 and Snf1 might be involved in the hierarchical regulatory network of genes under nitrogen catabolite repression. Dissection of this regulatory network could provide further insights to new targets for improving co-utilization of glucose and xylose.

Improvement of pyruvate production by Escherichia coli via pathway engineering and Tn5 transposon mediated mutagenesis / 生物工程学报

To develop a high-yield pyruvate strain, we first engineered a pyruvate-producing Escherichia coli KLPP from wild-type E. coli MG1655 by blocking the pathways for byproduct formation via gene knockout. Then, we built a library of mutant containing 7 197 monoclones by using the pUT Mini-Tn5 transposon vector for random mutagenesis with E. coli KLPP. We developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. After two-round screening we successfully obtained six mutants with increased pyruvate titer using this method, the titer of pyruvate was increased by 38%, 31%, 19%, 28%, 44% and 14%, respectively. The position of transposon insertion was determined by whole genome re-sequencing, and the gene locus possibly influencing pyruvate production was analyzed, which laid the foundation for subsequent strain improvement by metabolic engineering.

Construction and optimization of microbial cell factories for producing cis, cis-muconic acid / 生物工程学报

cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.

Construction and optimization of Escherichia coli for producing rhamnolipid biosurfactant / 生物工程学报

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.

Micro-droplet characterization and its application for amino acid detection in droplet microfluidic system / 生物工程学报

Recently, the droplet microfluidic system attracts interests due to its high throughput and low cost to detect and screen. The picoliter micro-droplets from droplet microfluidics are uniform with respect to the size and shape, and could be used as monodispensed micro-reactors for encapsulation and detection of single cell or its metabolites. Therefore, it is indispensable to characterize micro-droplet and its application from droplet microfluidic system. We first constructed the custom-designed droplet microfluidic system for generating micro-droplets, and then used the micro-droplets to encapsulate important amino acids such as glutamic acid, phenylalanine, tryptophan or tyrosine to test the droplets' properties, including the stability, diffusivity and bio-compatibility for investigating its application for amino acid detection and sorting. The custom-designed droplet microfluidic system could generate the uniformed micro-droplets with a controllable size between 20 to 50 microm. The micro-droplets could be stable for more than 20 h without cross-contamination or fusion each other. The throughput of detection and sorting of the system is about 600 micro-droplets per minute. This study provides a high-throughput platform for the analysis and screening of amino acid-producing microorganisms.

Progress in the thermophilic and alkalophilic xylanases / 生物工程学报

Xylanase is the key enzyme to degrade xylan that is a major component of hemicellulose. The enzyme has potential industrial applications in the food, feed, paper and flax degumming industries. The use of xylanases becomes more and more important in the paper industry for bleaching purposes. Xylanases used in the pulp bleaching process should be stable and active at high temperature and alkaline pH. Thermophilic and alkalophilic xylanases could be obtained by screening the wild type xylanases or engineering the mesophilic and neutral enzymes. In this paper, we reviewed recent progress of screening of the thermophilic and alkalophilic xylanases, molecular mechanism of thermal and alkaline adaptation and molecular engineering. Future research prospective was also discussed.

Improving 3-dehydroshikimate production by metabolically engineered Escherichia coli / 生物工程学报

In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS.