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Microbial fermentation is one of
the important industrial production methods.
Compared with general chemical synthesis and plant extraction, microbial fermentation conditions are mild, and it can use easy-to-obtain cheap substrates to propagate rapidly, with the advantages
of high yield, easy transformation, short cycle, easy extraction and purification.
With the development of medicine and food, the demand for microbial fermentation products is diversified, and the metabolic pathway of the required products is lacking in natural strains, or the metabolic pathway regulation is complex, and it is difficult to achieve excessive accumulation
of the required products.
As an important carrier of synthetic biology research, the emergence of synthetic biology technology has given new opportunities for the research of engineered bacteria, and the development of synthetic biotechnology has greatly improved the production capacity
of constructed engineered bacteria.
In synthetic biology research, microbes are often designed and engineered into cell factories for the production of
different products.
College of Food Science and Engineering, Jilin Agricultural University, Gao Xin, Liu Yuzhe, Min Weihong*, etc.
outlined synthetic biology technology, reviewed the transplantation or dynamic regulation of metabolic pathways with the help of editing tools and biological elements to construct engineering bacteria, and introduced the application
of constructed engineering bacteria in the production of amino acids, organic acids, aromatic compounds and sugars.
1.
Synthetic biology technology methods
Gene editing tools
The regularly spaced clusters of short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are emerging gene-editing tools for RNA-directed DNA recognition and editing
.
The development of this tool provides a new platform
for the construction of more efficient gene targeted modification techniques.
In 2013, Le Cong et al.
reported that the CRISPR/Cas system can simultaneously edit multiple sites in the mammalian genome, as an RNA-guided endonuclease system, which can directly target DNA sites
through nucleotide base pairing 。 Compared with traditional gene editing tools, CRISPR system as a new editing tool, with time-saving, easy to build, high precision characteristics, CRISPR system represented by the rapid development of new gene editing technology, in many biological fields have been widely used, become a popular tool for genome editing in recent years, has been widely used in gene knockout, gene silencing and gene activation, etc.
, greatly expanding the application scope of gene editing technology, as shown
in Figure 1.
Genome synthesis methods
With the rapid development of synthetic biology in the past 10 years, more and more scientific research teams are committed to using synthetic DNA to build complex gene systems
.
The artificial modification of the cell's own genome has naturally entered people's field of vision
.
In 2008, the Venter Institute constructed a complete copy of the 580 000 bp mycoplasma genome from chemically synthesized DNA; In 2016, the institute redesigned the previously synthesized mycoplasma genome to delete parts of genes and DNA identified as non-essential for growth, accounting for about half
of the entire genome.
The work does not encode any genes, and the part of the genome that remains is the same as the natural sequence, but it is currently the most artificially modified genome, and its gene content and layout are very different
from the natural genome.
2.
Design theory of synthetic biology
Bioelement design and dynamic regulation
Basic element design: Biological elements are the basic building blocks with the most basic biological functions in living organisms, such as regulatory elements that regulate gene expression, including promoters, terminators, ribosome binding sites (RBS), and structural elements with specific functions (such as enzyme genes in the natural product synthesis pathway).
In recent years, with the continuous improvement of the understanding of the core elements of promoter sequences and upstream activation sequences, the development and application of artificial heterozygous promoters are developing
rapidly.
In the future, it is expected that promoters with shorter sequences and stronger performance will be obtained, which will facilitate the more effective regulation
of complex pathways introduced in model bacteria.
The method of library construction to design regulatory elements such as promoters or RBS is very effective, but library construction and component screening are inefficient, not suitable for artificial biological system construction that requires a large number of regulatory elements, and many components cannot be quickly library screened
.
Therefore, it is important to
establish a complete set of promoter strength prediction models.
Sensor and switch design: Dynamic regulation with the help of biosensors can replace conventional gene knockout, thereby solving the problem
of poor bacterial growth affecting yield due to gene knockout.
At present, biosensors mainly include fluorescence resonance energy transfer sensors, ribose switch sensors and transcription factor sensors
.
Fluorescence resonance energy transfer sensors are often used in the field of food safety testing due to their simple operation, high sensitivity and fast reaction speed, such as the detection
of ochratoxin A.
The riboswitch responds more quickly to ligands, has a simple structure and is easy to modify, and is more suitable for the dynamic regulation
of gene expression.
Wang Xing designed a sensing system with high sensitivity to the concentration of isopropyl galactoside, including a signaling molecule LuxI protein expression element and a sensing element
that can accept the stimulation of signal molecules to produce green fluorescent protein.
Transcription factor sensors control downstream gene expression by recognizing and binding elements of gene promoter regions with transcription factors, and their properties that regulate gene expression are suitable for setting up cell factories, such as screening highly active enzymes and high-yielding bacteria, and dynamically regulating protein expression
.
Protein design
Based on current levels of understanding, the complete de novo design of functional biological components is a difficult challenge, especially in amino
acid sequencing and high-level structural determination.
Many protein elements lack high-throughput models to rapidly perform library screening to obtain mutants
with the desired function.
Therefore, according to the special relationship between the mastered component sequence and the function, the computer model is established, the key sites of the component are transformed, and the rational design of the component with the expected function and control characteristics is an important direction
of component design 。 In the design of protein elements, the DNA sequence encoding the biological element can be replaced based on the relationship between the known element structure and function, or the unnatural amino acid sequence can be introduced to obtain the transformed protein element.
Secondly, according to large-scale directional evolution screening, random mutations can be introduced into the DNA encoding biological elements, and large-scale screening can be carried out with appropriate screening methods to obtain the desired protein biological elements.
Computational simulations can also be used to design and synthesize topologies in proteins, and proteins with special structures can be synthesized from de novo
.
Synthetic circuit and synthetic network design
Based on synthetic biological elements, multiple elements are connected in series or parallel to form a synthetic gene circuit
with information processing ability and intervention in the function of living organisms.
The design of synthetic gene circuits first requires the construction of basic functional components, such as toggle switches, sensors, etc.
, and then component assembly to build complex functional circuits
.
The artificial synthesis of gene circuits helps to delete the genome of the bacterium, and genome simplification can improve people's understanding of genome function and create streamlined and efficient engineered bacteria, such as deleting duplicate parts of the bacterial metabolic regulatory network, stabilizing the insertion of foreign genes, optimizing the bacterial metabolic pathway, and enhancing the use
of substances and energy by the bacterium.
In recent years, some progress has been made in the design of synthetic gene circuits, and scientists have constructed a number of functional components
such as gene sensors and regulatory elements with the required strength, gene expression dynamic controllers, and biological triggers.
Based on current research, synthetic gene circuits constructed within a single cell are difficult to carry a large number of artificial additional elements
.
Competition and noise in gene expression limit the scale of gene circuits, and reducing noise often requires increasing gene expression intensity, straining
limited resources and energy allocation within cells.
3.
Application of synthetic biology to construct microbial engineering bacteria
amino acid
The development of synthetic biology technology has a great role in promoting the amino acid fermentation production industry, which can not only improve the yield of traditional amino acid products, but also obtain engineering bacteria
that produce special amino acid products.
Zhou Libang et al.
used lysine ribose switches and intracellular L-lysine as signals to control the necessary metabolic bypass that competes with lysine biosynthesis, which reduces the impact
on bacterial growth and metabolism compared with the traditional one-size-fits-all modification methods.
Long Mengfei designed a gene regulatory circuit that can dynamically regulate the activity of α-ketoglutarate dehydrogenase, and used the regulatory system to down-regulate the key enzyme activities of the branch, which changed the host progenome by a small extent compared with the traditional knockout of the branch, and improved the yield
of trans-4-hydroxy-L-proline.
Aromatic compounds
Aromatic compounds containing alternating unsaturated rings with double and single bonds are widely used in industry, for example as additives in pharmaceuticals, agrochemicals, food, feed and cosmetics
.
Aromatic compounds are mainly synthesized by chemical synthesis and can also be obtained
from plants by extraction.
Due to a series of resource and environmental issues, biotechnology to produce aromatics from renewable biomass is receiving increasing attention
.
Many microorganisms are capable of catabolizing too many aromatic compounds, and intercepting these metabolic pathways may lead to biotechnology production of value-added aromatic compounds, and researchers have focused on the shikimic acid pathway, an important pathway
for the production of aromatic amino acids and a variety of other aromatic compounds.
In the shikimic acid pathway, the final product 4-hydroxybenzoic acid (4HBA) is a raw material
with valuable use in antiseptic and sterilization.
saccharide
Glucosamine (GlcN) is often used as a dietary supplement, and GlcN and its derivative N-acetylglucosamine (GlcNAc) have the effect of controlling pain, improving function and delaying joint structural changes, and are widely used
in the pharmaceutical field.
Liu Yanfeng co-expressed D-glucosamine-6-phosphate synthase and D-glucosamine-6-phosphate acetylase in Bacillus subtilis, successfully constructed the GlcNAc synthesis pathway, and realized the accumulation
of GlcNAc in Bacillus subtilis.
On the basis of this recombinant Bacillus subtilis, the key enzymes of the GlcNAc synthesis pathway were regulated, the double promoter system was used to optimize the expression of key enzymes in the GlcNAc synthesis pathway, and the rigorous promoter PxylA regulated the expression of D-glucosamine-6-phosphate synthase, and inhibited the branching metabolic pathway to obtain high-yield recombinant strains
.
Organic acids
Fermentation method is used in the production of a variety of organic acids, organic acids in the field of medicine is widely demanded, so organic acid production has gradually become an important part of
the fermentation industry.
Shikimic acid is a key intermediate in the synthesis of the antiviral drug oseltamivir, its derivatives have antitumor, prevent thrombosis and other effects, can be obtained by chemical synthesis, microbial fermentation and extraction from certain plants, due to the high cost of plant extraction, the production of shikimic acid in recombinant microorganisms by fermentation may become the established preferred pathway
.
Hou Jianfen et al.
used growth-dependent promoters and electron removal to construct a dynamic molecular switch, which separated cell growth from shikimic acid synthesis, and generated shikimic acid 14.
33 g/L
after fermentation for 72 h.
Liu Chang et al.
constructed a library of adaptability-enhancing 3-dehydroquinic acid dehydratase elements based on the preference for genetic codon use in Escherichia coli and Corynebacterium glutamate, customized new DNA sequences for synthetic biology applications, and obtained artificial modules
of shikimic acid pathway that can be used to design metabolic engineering and synthetic biology.
Natural products
Natural products have complex and diverse structures and multiple activities, but their natural synthetic pathways and metabolic regulation are complex, and it is necessary to systematically design and modify the host synthetic pathways to improve the biosynthesis efficiency
of natural products.
With the increasing maturity of synthetic biology technology, heterologous expression has been widely used in the production of
natural products.
Liang Rong et al.
used the eukaryotic expression system of Pichia pichia to achieve heterologous expression of Arabidopsis thaliana AtPOFUT1 protein, and preliminary detection confirmed that the recombinant protein had O-fucosyltransferase activity
.
Padadon et al.
overexpressed plant dehydrogenase and cytochrome P450 by reengineering the synthetic pathway, so that the fermentation yield of artemisinic acid in Saccharomyces cerevisiae reached 25 g/L, which provided the possibility
for the mass production of precursors for the chemical synthesis of artemisinin.
Conclusion
The research of synthetic biology related technologies provides strong technical support for the construction and production of engineering strains of expected compounds, which is an important directionof research in the field of fermentation industry.
The use of synthetic biology technology to construct high-efficiency microbial engineering bacteria can maintain normal growth of bacteria, so that industrial microorganisms can improve the existing production capacity and physiological performance
on the basis of stable metabolism.
The application of synthetic biology tools to directed evolution can shorten the directional evolution cycle of engineered bacteria, increase the efficiency of mutant screening, apply it to metabolic engineering, and rapidly improve product diversity
by dynamically controlling the expression of each complex pathway under the premise of engineering the biological system as a whole.
The research on fermentation of engineering bacteria, represented by the development of functional components such as amino acids and GlcN, has made remarkable progress
.
On the basis of theoretical research, technical exploration and reference to typical synthetic circuits, further broadening the target range of synthesis, constructing intelligent engineering bacteria and realizing large-scale production are important directions
of synthetic biology technology in the future.
About the corresponding author
Prof.
Min Weihong, Dean of College of Food Science and Engineering, Jilin Agricultural University, second-level professor, doctoral supervisor
.
Excellent teacher of Jilin Province, top-notch innovative second-level talent of Jilin Province, young and middle-aged expert with outstanding contributions in Jilin Province, winner of Jilin Youth Science and Technology Award, outstanding talent of science and technology in the new century of colleges and universities in Jilin Province, good person of Jilin Province - the most beautiful teacher team member, executive director
of the Agricultural Products Processing and Storage Branch of the Chinese Society of Agricultural Engineering 。 Jilin Agricultural University light industry technology and engineering first-level discipline leader, Jilin Agricultural University teaching master, Jilin Agricultural University food science and engineering dean, engaged in enzyme molecular transformation and food nutrition molecular regulation research, in AK directional evolution and transformation, active peptide to reduce oxidative stress on nerve cell protection and improve learning and memory ability and other
aspects of breakthroughs 。 In the past five years, he has presided over 11 national, provincial and ministerial projects such as the general projects of the National Natural Science Foundation of China, the national "863" plan, the national "Twelfth Five-Year" science and technology support project, the agricultural achievement transformation fund project of the Ministry of Science and Technology, and the key science and technology projects of the Science and Technology Department of Jilin Province, with a cumulative fund of 15 million yuan; He has won the second prize of National Science and Technology Progress Award and the first prize of China Agricultural Science and Technology Achievement Award, published 19 SCI papers (6 top journals), 15 EI papers and 4 invention patents in foreign journals such as Food Chemistry and Journal of Agricultural and Food Chemistry
.
He supervised 87 doctoral and master students, including 3 international students
.
This article "Research Progress on the Construction of Microbial Engineering Bacteria in Synthetic Biology" is from Food Science, Vol.
43, No.
15, pp.
256-264, 2022, authors: Gao Xin, Liu Yuzhe, Jiang Zeyuan, Zeng Qi, Liu Shimeng, Liu Xiaoting, Min Weihong
.
DOI:10.
7506/spkx1002-6630-20210831-414
。 Click to view information about
the article.
