Adaptive evolution of microorganisms based on industrial environmental perturbations / 生物工程学报

The development of synthetic biology has greatly promoted the construction of microbial cell factories, providing an important strategy for green and efficient chemical production. However, the bottleneck of poor tolerance to harsh industrial environments has become the key factor hampering the productivity of microbial cells. Adaptive evolution is an important method to domesticate microorganisms for a certain period by applying targeted selection pressure to obtain desired phenotypic or physiological properties that are adapted to a specific environment. Recently, with the development of technologies such as microfluidics, biosensors, and omics analysis, adaptive evolution has laid the foundation for efficient productivity of microbial cell factories. Herein, we discuss the key technologies of adaptive evolution and their important applications in improvement of environmental tolerance and production efficiency of microbial cell factories. Moreover, we looked forward to the prospects of adaptive evolution to realize industrial production by microbial cell factories.

Chromosomal integration of large DNA fragments in microorganisms: a review / 生物工程学报

The modern bio-fermentation industry requires design and creation of efficient microbial cell factories for directed conversion of raw materials to target products. The main criteria for assessing the performance of microbial cell factories are their product synthesis capacity and stability. Due to the deficiencies of plasmids in gene expression such as instability and being easy to lose, integration of genes into chromosome is often a better choice for stable expression in microbial hosts. To this end, chromosomal gene integration technology has received much attention and has developed rapidly. In this review, we summarize the recent research progresses of chromosomal integration of large DNA fragments in microorganisms, illustrate the principles and features of various technologies, highlight the opportunity brought by the CRISPR-associated transposon systems, and prospect future research direction of this technology.

Advances in using adaptive laboratory evolution technology for engineering of photosynthetic cyanobacteria / 生物工程学报

Cyanobacteria are the only prokaryotes capable of oxygenic photosynthesis, which have potential to serve as "autotrophic cell factories". However, the synthesis of biofuels and chemicals using cyanobacteria as chassis are suffered from poor stress tolerance and low yield, resulting in low economic feasibility for industrial production. Thus, it's urgent to construct new cyanobacterial chassis by means of synthetic biology. In recent years, adaptive laboratory evolution (ALE) has made great achievements in chassis engineering, including optimizing growth rate, increasing tolerance, enhancing substrate utilization and increasing product yield. ALE has also made some progress in improving the tolerance of cyanobacteria to high light intensity, heavy metal ions, high concentrations of salt and organic solvents. However, the engineering efficiency of ALE strategy in cyanobacteria is generally low, and the molecular mechanisms underpinning the tolerance to various stresses have not been fully elucidated. To this end, this review summarizes the ALE-associated technical strategies and their applications in cyanobacteria chassis engineering, following by discussing how to construct larger ALE mutation library, increase mutation frequency of strains and shorten evolution time. Moreover, exploration of the construction principles and strategies for constructing multi-stress tolerant cyanobacteria, and efficient analysis the mutant libraries of evolved strains as well as construction of strains with high yield and strong robustness are discussed, with the aim to facilitate the engineering of cyanobacteria chassis and the application of engineered cyanobacteria in the future.

Biosynthesis of natural products by non-conventional yeasts / 生物工程学报

Non-conventional yeasts such as Yarrowia lipolytica, Pichia pastoris, Kluyveromyces marxianus, Rhodosporidium toruloides and Hansenula polymorpha have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization spectrum, strong tolerance to environmental stresses and other merits. With the development of synthetic biology and gene editing technology, metabolic engineering tools and strategies for non-conventional yeasts are expanding. This review introduces the physiological characteristics, tool development and current application of several representative non-conventional yeasts, and summarizes the metabolic engineering strategies commonly used in the improvement of natural products biosynthesis. We also discuss the strengths and weaknesses of non-conventional yeasts as natural products cell factories at current stage, and prospects future research and development trends.

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.

Metabolic regulation in constructing microbial cell factories / 生物工程学报

The regulation of the expression of genes involved in metabolic pathways, termed as metabolic regulation, is vital to construct efficient microbial cell factories. With the continuous breakthroughs in synthetic biology, the mining and artificial design of high-quality regulatory elements have substantially improved our ability to modify and regulate cellular metabolic networks and its activities. The research on metabolic regulation has also evolved from the static regulation of single genes to the intelligent and precise dynamic regulation at the systems level. This review briefly summarizes the advances of metabolic regulation technologies in the past 30 years.

Thirty years development of metabolic engineering: a review / 生物工程学报

Since its establishment 30 years ago, the discipline of metabolic engineering has developed rapidly based on its deep integration with molecular biology, systems biology and synthetic biology successively, which has greatly contributed to advancing and upgrading biotechnology industry. This review firstly analyzes the current status of academic research and China's competence in the area of metabolic engineering according to the data of papers published in SCI-indexed journals in the past 30 years. Subsequently, the article summarizes the development of systems biology methods and enabling technologies of synthetic biology and their applications in metabolic engineering in the past 10 years. Finally, the major challenges and future perspectives for the development of metabolic engineering are briefly discussed.

Application of chronological lifespan in the construction of Escherichia coli cell factories / 生物工程学报

Microbial cell factories capable of producing valuable chemicals from renewable feedstocks provide a promising alternative towards sustainability. However, environmental stress remarkably affects the performance of microbial cell factories. By extending the chronological lifespan of microbial cells, the performance of microbial cell factories can be greatly improved. Firstly, an evaluation system for chronological lifespan and semi-chronological lifespan was established based on the changes in survival rates. Secondly, the addition of anti-aging drugs such as cysteine, carnosine, aminoguanidine and glucosamine increased the chronological lifespan of E. coli by 80%, 80%, 50% and 120%, respectively. Finally, we demonstrated that extending the chronological lifespan of E. coli increased the yield of metabolites produced by E. coli cell factories with endogenous (lactic acid and pyruvic acid) or exogenous (malic acid) metabolic pathway by 30.0%, 25.0%, and 27.0%, respectively. The strategy of extending chronological lifespan of E. coli provides a potential approach for enhancing the performance of microbial cell factories.

Progress and perspective on development of non-model industrial bacteria as chassis cells for biochemical production in the synthetic biology era / 生物工程学报

The development and implement of microbial chassis cells can provide excellent cell factories for diverse industrial applications, which help achieve the goal of environmental protection and sustainable bioeconomy. The synthetic biology strategy of Design-Build-Test-Learn (DBTL) plays a crucial role on rational and/or semi-rational construction or modification of chassis cells to achieve the goals of "Building to Understand" and "Building for Applications". In this review, we briefly comment on the technical development of the DBTL cycle and the research progress of a few model microorganisms. We mainly focuse on non-model bacterial cell factories with potential industrial applications, which possess unique physiological and biochemical characteristics, capabilities of utilizing one-carbon compounds or of producing platform compounds efficiently. We also propose strategies for the efficient and effective construction and application of synthetic microbial cell factories securely in the synthetic biology era, which are to discover and integrate the advantages of model and non-model industrial microorganisms, to develop and deploy intelligent automated equipment for cost-effective high-throughput screening and characterization of chassis cells as well as big-data platforms for storing, retrieving, analyzing, simulating, integrating, and visualizing omics datasets at both molecular and phenotypic levels, so that we can build both high-quality digital cell models and optimized chassis cells to guide the rational design and construction of microbial cell factories for diverse industrial applications.

Progress in biological utilization of formic acid / 生物工程学报

The use of microbial cell factories to achieve efficient conversion of raw materials and synthesis of target substances is one of the important research directions of synthetic biology. Traditional industrial microorganisms have mainly used sugar-based raw materials as fermentation substrates. How to adopt cheaper carbon resources and realize their efficient use has been widely concerned. Formic acid is an important organic one-carbon source and widely used in industrial manufacturing of pesticides, leather, dyes, medicine and rubber. In recent years, due to the demand fluctuation in downstream industries, formic acid production is facing the dilemma of overcapacity, and therefore, requiring new conversion paths for expansion and extension of the related industrial chain. Biological route is one of the important options. However, natural formate-utilizing microorganisms generally grow slowly when metabolizing formic acid, and moreover, are difficult to be artificially modified by the absence of effective genetic tools. Construction of non-natural formate-utilizing microorganisms is another alternative strategy, but still in its infancy and has a huge space for further improvements. Here, we briefly summarize the recent research progress of biological utilization of formic acid, and also propose the future research focus and direction.

Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-II / 药学学报

Dammarenediol-Ⅱ is an important precursor in the biosynthesis pathway of ginsenosides which are the main active components of Panax quinquefolius and Panax ginseng. For constructing a dammarenediol- Ⅱ-producing cell factory, the triterpenoid precursors of yeast are improved significantly by the modular pathway engineering strategy on the basis of an MVA optimized strain. The strain overexpressing Salvia miltiorrhiza SmFPS and Arabidopsis thaliana AtSQS2 could yield 67.4 mg·g−1 squalene, accounting for about 6.74% of cell dry weight. In our further work, an Arabidopsis thaliana 2,3-oxidosqualene synthase AtSQE2 was found to be able to increase the downstream lanosterol yield by 22-fold, reaching 47.9 mg·g−1. Then, regulating dammarenediol-Ⅱ synthase gene expression, using anti-sense RNA technology for regulation of ERG7 in the ergosterol pathway, and optimizing fermentation process were successively performed. Finally, the synthesis flux of triterpenes was increased to 10 g·L−1 for the first time, and we constructed an efficient cell factory that can produce 15 g·L−1 dammarenediol-Ⅱ, which lays a solid foundation of industrial synthesis of dammarane-type ginsenosides.