Poly-γ-glutamic acid (γ-PGA) is a kind of poly-amino acid formed by polymerization of L-glutamic acid (L-Glu) and / or D-glutamic acid (D-Glu), and is widely used in cosmetics, medicine and other fields. The stereochemical composition of its monomer often affects the properties and applications of the product. Therefore, it is of great significance to control the monomer ratio of D-Glu/L-Glu (D/L monomer ratio) in γ-PGA. In our previous study, Corynebacterium glutamicum was used as the chassis to overexpress γ-PGA synthetase from Bacillus licheniformis, and synthesize γ-PGA with L-Glu as the main component. On this basis, different concentrations of D-Glu were added exogenously to synthesize γ-PGA with D-Glu accounting for 15.71% ~ 33.52%. Then, the glutamate racemase from Bacillus subtilis was overexpressed in the recombinant bacteria, and three RBS of different strengths were used to regulate its expression level, however, and γ-PGA with D-Glu accounting for a narrow range (30.82%~34.59%) was synthesized. Subsequently, four different strength promoters were used to regulate the expression level of glutamate racemase, and γ-PGA with D-Glu accounting for 32.71%~52.53% was synthesized. A rational strategy for regulating the D/L monomer ratio of γ-PGA was proposed, and γ-PGA with a D-Glu ratio of 2.90%~52.53% was synthesized, which laid the foundation for the efficient synthesis of γ-PGA with different D/L monomer ratios.
Objective: Indole-3-acetic acid (IAA) was produced from tryptophan in the metabolically engineered E. coli MG1655 using whole-cell catalysis. Methods: Two novel IAA biosynthetic pathways, the indole-3-acetamide (IAM) pathway and the tryptamine (TRP) pathway, were constructed in E. coli MG1655. Results: The IAM pathway involves two enzymes, tryptophan-2- monooxygenase (IAAM) and amidase (AMI1). 2g/L tryptophan as a substrate was used by the constructed recombinant E. coli strain TPA-4. TPA-4 can produce 0.803g/L of IAA; however, in the strain MPA-3 that was knocked out the gene tnaA which divert flux from tryptophan synthesis, the yield of IAA reached 1.43g/L, an increase of 78% compared with the control. The second TRP pathway biosynthesis of IAA involves three enzymes: L-tryptophan decarboxylase (TDC), diamine oxidase (AOC1) and indole-3-acetaldehyde dehydrogenase (IAD1). The recombinant E.coli TPTA-2 that included the TRP pathway can only synthesize 13.0mg/L IAA with 2g/L tryptophan as substrate. In the strain MPTA-3 with disruption of tnaA gene, 21.0mg/L of IAA was finally produced, and the yield increased by 61.5%. Conclusion: It is the first report to realize production of IAA using the metabolically engineered E. coli through the IAM pathway and TRP pathway via whole-cell catalysis. IAA production from the IAM pathway is relatively higher, and it probably has an industrial application prospect.
As a phase sensitive fluorescent probe, Di-4-ANEPPDHQ can specifically label the ordered phase and disordered phase of membrane. Therefore, in theory, the probe can be used to quantitatively image the order of cell membrane. By combining Di-4-ANEPPDHQ and laser scanning confocal microscopy, the ordered phase and disordered phase live-cell imaging of a variety of representative industrial model microorganisms were carried out. Combined with the statistical comparison of polarity normalization values, the quantitative analysis of the cell membrane ordering of the above industrial model microorganisms is finally realized. The above study provides an intuitive and rapid detection method of living cells for cell membrane engineering.
Lanthipeptides are a major class of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with diverse molecular structures and biological activities. New lanthipeptides obtained by genome mining and engineering are an important source of drug leads. Lanthipeptides are particularly amenable to bioengineering because their precursor peptides are encoded by genes and biosynthetic enzymes often exhibit high promiscuity, which is helpful for the efficient construction of lanthipeptides derivatives. This paper reviews the recent advances in high-throughput creation and screening of lanthipeptide derivatives. For mutant library creation, we discuss the introduction of noncanonical amino acids (ncAAs), combinatorial biosynthesis, and chimeric-leader approach for creating hybrid RiPPs. Then, we introduce large-scale structural and activity screening of lanthipeptide mutants assisted by cell surface display, reverse two-hybrid system, cellular autolysis, cell-free system, and microfluidics. Finally, we present future perspectives on the use of synthetic biology automation to streamline lanthipeptide bioengineering.
Directed evolution provides a simple and high-efficiency tool for the development of synthetic biology, especially in the chemical synthesis and medicine. However, the traditional directed evolution technique has the problems of cumbersome operation, time-consuming and low-efficiency, which cannot satisfy the construction and screening of mass mutant libraries. In recent years, a technique about in vivo continuous directed evolution that seamlessly integrates mutation, translation (if the evolving molecules are not genes themselves), screening and replication processes into an uninterrupted cycle has emerged, which makes breakthrough in phage, bacteria and eukaryotic cells, greatly facilitating technological innovation of directed evolution. Simultaneously, with the development of in vivo continuous evolution technology, screening methods and evolutionary devices are also constantly improved. Here, this review is done to expound the latest research progress on continuous directed evolution techniques, screening methods and devices, and discuss the current challenges and opportunities.
Lignocellulosic biomass is considered an important and sustainable renewable energy source, which contains cellulose as the main component of lignocellulosic biomass. Cellulase is a group of enzymes that can decompose cellulose into glucose. Various microorganisms including fungi, bacteria, actinomycetes and yeasts are known to produce cellulase. Among them, Trichoderma reesei is one of the most widely used cellulolytic organisms that can produce a large amount of intact extracellular cellulase and hemicellulase to degrade lignocellulose. Therefore, T. reesei has become an important host microorganism for the production of commercial enzymes in the field of biotechnology. This article introduces the mechanism of cellulase, and summarizes the development status and the latest research progress of cellulase production by T. reesei. The influences of cellulase production technology including fermentation conditions and cellulase inducers, and the progress of different molecular strategies (such as mutagenesis and genetic modification) on constructing T. reesei with high cellulase-producing abilities were reviewed. In addition, the bottlenecks related to cellulase production are also discussed, in order to provide a more economical production process to widely apply cellulase in industrial production.
With the development of industrialization, heavy metal pollution has become a severe environmental hazard, in particular its pollution to water systems and consequently endangering human health, making its remediation an urgent issue. Compared with traditional physical and chemical remediation, bioremediation is green and sustainable. Microorganisms and microbial biofilms play an important role in bioremediation due to their rapid growth, intrinsic nature for dynamic adaption to environments and consequent tolerance to environmental stresses. Synthetic biology has provided a methodology for engineering and reprograming microorganisms and biofilms with robustness for more efficient degradation of environmental pollutants including heavy metals. This review summarizes the current status of heavy metal pollution, the bioremediation mechanism and research progress of microbial remediation, with a focus on the development of functional biofilms engineered by synthetic biology and their applications in bioremediation for heavy metal Pb, Hg, Cd and others. At the end, this review highlights the perspectives for bioremediation of heavy metal pollution.
Human epidermal growth factor (hEGF) is a kind of protein isolated from the human body. It can stimulate the cell growth and thus has a wide range of clinical applications. The use of genetic engineering to produce hEGF has great economic prospects due to its limited natural source and high cost of chemical synthesis. Yeast expression system, as a typical eukaryotic expression system, can perform post-translational modifications, such as glycosylation, disulfide bond formation, etc., and can effectively secrete the proteins outside of the cell so that the recombinant protein can be separated from the extracellular medium easily. Therefore, yeast is especially favored in the research of producing recombinant hEGF. This article reviews the progress of producing hEGF in various yeast expression systems, including Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, etc., and on this base, their pros and cons were compared. The future prospects of using yeast expression to produce hEGF are discussed in light of the current progress, challenges, and trends in this field. Guidelines for future studies are finally proposed.
Ginsenosides are the main active substances of traditional medicinal plants and are widely used in the fields of medicine, health care products, nutritional products and cosmetics. In particular, rare ginsenosides have a variety of biological activities, such as significant anti-tumor activity and nervous system protection and liver protection function. However, the low yield of rare ginsenosides in plants seriously affects its development and utilization. Since the end of the 20th century, with the continuous development of biotechnology and genome sequencing, the problem of low production of ginsenosides can be solved by heterologous synthesis of ginsenosides in microorganisms. Therefore, it has attracted more and more attentions such as constructing an artificial synthetic system of ginsenosides, regulating ginsenoside biosynthesis strategies, and increasing the yield of saponinsin microorganisms. We reviewed the construction of heterologous synthetic pathways of ginsenosides and the research progress of their biosynthetic regulation strategies in this review. Finally, we summarized and prospected the future research directions of ginsenosides synthesis process and regulation process in microorganisms.
Avilamycin, an oligosaccharide antibiotic with strong antibacterial effect to Gram-positive intestinal pathogenic bacteria, has been widely used in livestock and poultry breeding, such as broiler chickens and piglets, as a new type of feed additive. The antibacterial mechanism, structural modification, high yield strain breeding and fermentation optimization of avilamycin were briefly reviewed and the gene clusters, synthetic pathways and transcriptional regulation mechanisms of avilamycin were highlighted, based on research progress in the past five years, in this work. Further, the strategies of genetic engineering to improve the yield of avilamycin were discussed, which could provide some references for the efficient synthesis of avilamycin and the construction of industrial high-yielding engineered strains.
Pyrroloquinoline quinone (PQQ) is a ribosomally synthesized and post-translationally modified peptide that has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It is widely distributed in organisms and has physiological functions such as antioxidation. It has broad application prospects in the fields of medicine, food and cosmetics. However, the lack of an efficient microbial cell factory limits the industrial production efficiency of PQQ. In this paper, we reviewed the current research on the biosynthesis pathway of PQQ, the structures of key enzymes, as well as metabolic engineering strategies for the construction of high-yielding strains. These advances will provide a basis for finally achieving stable PQQ biosynthesis in microbial cell factories.
