This article reviews the review articles and research papers related to biomanufacturing driven by engineered organisms published in the Chinese Journal of Biotechnology from 2023 to 2024. The content covers 26 aspects, including chassis cells; gene (genome) editing; facilities, tools and methods; biosensors; protein design and engineering; peptides and proteins; screening, expression, characterization and modification of enzymes; biocatalysis; bioactive substances; plant natural products; microbial natural products; development of microbial resources and biopesticides; steroidal compounds; amino acids and their derivatives; vitamins and their derivatives; nucleosides; sugars, sugar alcohols, oligosaccharides, polysaccharides and glycolipids; organic acids and monomers of bio-based materials; biodegradation of polymeric materials and biodegradable materials; intestinal microorganisms, live bacterial drugs and synthetic microbiomes; microbial stress resistance engineering; biodegradation and conversion utilization of lignocellulose; C1 biotechnology; bioelectron transfer and biooxidation-reduction; biotechnological environmental protection; risks and regulation of biomanufacturing driven by engineered organisms, with hundreds of technologies and products commented. It is expected to provide a reference for readers to understand the latest progress in research, development and commercialization related to biomanufacturing driven by engineered organisms.
Biotechnology/methods* ; Gene Editing ; Genetic Engineering ; Metabolic Engineering ; Protein Engineering ; Biosensing Techniques

L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.
Citrulline/biosynthesis* ; Escherichia coli/genetics* ; Metabolic Engineering/methods* ; Fermentation ; NADP/biosynthesis*

O-acetyl-L-homoserine (OAH) is a promising platform compound for the production of L-methionine and other valuable compounds, while its low yield and low conversion rate limit the industrial application. To solve these problems, we constructed a strain for high OAH production with the previously constructed L-homoserine producer Escherichia coli HS33 as the chassis by systematic metabolic engineering. Firstly, PEP accumulation, pyruvate utilization, and OAH synthesis pathway (overexpressing aspB, aspA, and thrA^C1034T) were enhanced to obtain an initial strain accumulating 13.37 g/L OAH. Subsequently, the co-factor synthesis genes were integrated to supply reducing power and energy, which increased the yield to 15.79 g/L. The OAH yield of the engineered strain OAH28 was further increased to 17.49 g/L by strengthening the acetic acid reuse pathway, improving the supply of acetyl-CoA, and regulating the expression of MetX from different sources. Finally, in a 5 L fermenter, OAH28 achieved an OAH titer of 47.12 g/L, with a glucose conversion rate of 32% and productivity of 0.59 g/(L·h). The results lay a foundation for increasing the OAH production by metabolic engineering and give insights into the industrial production of OAH.
Metabolic Engineering/methods* ; Escherichia coli/genetics* ; Homoserine/biosynthesis* ; Fermentation

The efficient production of L-glutamate is dependent on the product's rapid efflux, hence researchers have recently concentrated on artificially modifying its transport system and cell membrane wall structure. Considering the unique composition and structure of the cell wall of Corynebacterium glutamicum, we investigated the effects of CmpLs on L-glutamate synthesis and transport in SCgGC7, a constitutive L-glutamate efflux strain. First, the knockout strains of CmpLs were constructed, and it was confirmed that the deletion of CmpL1 and CmpL4 significantly improved the performance of L-glutamate producers. Next, temperature-sensitive L-glutamate fermentation with the CmpL1 and CmpL4 knockout strains were carried out in 5 L bioreactors, where the knockout strains showcased temperature-sensitive characteristics and enhanced capacities for L-glutamate production under high temperatures. Notably, the CmpL1 knockout strain outperformed the control strain in terms of L-glutamate production, showing production and yield increases of 69.2% and 55.3%, respectively. Finally, the intracellular and extracellular metabolites collected at the end of the fermentation process were analyzed. The modification of CmpLs greatly improved the L-glutamate excretion and metabolic flux for both L-glutamate production and transport. Additionally, the CmpL1 knockout strain showed decreased accumulation of downstream metabolites of L-glutamate and intermediate metabolites of tricarboxylic acid (TCA) cycle, which were consistent with its high L-glutamate biosynthesis capacity. In addition to offering an ideal target for improving the stability and performance of the industrial strains for L-glutamate production, the functional complementarity and redundancy of CmpLs provide a novel target and method for improving the transport of other metabolites by modification of the cell membrane and cell wall structures in C. glutamicum.
Corynebacterium glutamicum/genetics* ; Glutamic Acid/biosynthesis* ; Fermentation ; Metabolic Engineering ; Bacterial Proteins/metabolism* ; Bioreactors/microbiology* ; Gene Knockout Techniques

Transcriptional regulation based on transcription factors is an effective regulatory method widely used in microbial cell factories. Currently, few naturally transcriptional regulatory elements have been discovered from Saccharomyces cerevisiae and applied. Moreover, the discovered elements cannot meet the demand for specific metabolic regulation of exogenous compounds due to the high background expression or narrow dynamic ranges. There are abundant transcriptional regulatory elements in plants. However, the sequences and functions of most elements have not been fully characterized and optimized. Particularly, the applications of these elements in microbial cell factories are still in the infancy stage. In this study, natural regulatory elements from Medicago truncatula were selected, including the transcription factors MtTASR2 and MtTASR3, along with their associated promoter P_roHMGR1, for functional characterization and engineering modification. We constructed an inducible transcriptional regulation tool and applied it in the regulation of heterologous β-carotene synthesis in S. cerevisiae, which increased the β-carotene production by 7.31 folds compared with the original strain. This study demonstrates that plant-derived transcriptional regulatory elements can be used to regulate the expression of multiple genes in S. cerevisiae, providing new strategies and ideas for the specific regulation and application of these elements in microbial cell factories.
Medicago truncatula/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Transcription Factors/genetics* ; beta Carotene/biosynthesis* ; Promoter Regions, Genetic/genetics* ; Gene Expression Regulation, Plant ; Metabolic Engineering/methods* ; Regulatory Elements, Transcriptional/genetics* ; Plant Proteins/genetics*

Wickerhamomyces ciferrii (W.c), an unconventional heterothallic yeast species, is renowned for its high production of tetraacetyl phytosphingosine (TAPS). Due to its excellent performance in TAPS production, this study aimed to construct a genetic operating system of W.c to enhance the production of TAPS and to screen high-yielding strains by mutagenesis and genetic engineering, thus laying the foundation for further development of industrial production of sphingolipid metabolites. In this study, we selected two autonomous replication elements (CEN, 2μ) and mined 11 endogenous promoter elements to establish a genetic operating system in W. ciferrii. The overexpression of Syr2 and Lcb2 in the sphingolipid metabolism pathway significantly increased the production of TAPS. Meanwhile, we established a method for the identification of haploid mating types of W. ciferrii by combining RT-PCR and flow cytometry. Five strains of W. ciferrii with different mating types constructed from the standard diploid W. ciferrii ATCC 14091 were screened out. A-type haploid W.c 140 showcased the highest production of TAPS with a yield of 4.74 mg/g and a titer of 32.61 mg/L. Mutant strains W.c 140-A9 and W.c 140-A11 were induced by atmospheric pressure room temperature plasma mutagenesis. The recombinant strains W.c 140 OELcb2 and W.c 140 OESyr2 with overexpression were constructed with the genetic operating system established in this study. The TAPS yields of the mutant strains increased by 61.39% and 67.09%, respectively, compared with that of starting strain W.c 140. The recombinant strains cultured in the LCBNB medium achieved yields of 10.60 mg/g and 12.14 mg/g, respectively, representing 2.24 and 2.56 times of that in strain W.c 140. Moreover, the yields of the two recombinant strains were significantly higher than that of the diploid strain ATCC 14091. The genetic operating system and the haploid strain W.c 140 established in this study provide a basis for the subsequent establishment of genetic engineering tools for W. ciferrii.
Sphingosine/genetics* ; Saccharomycetales/metabolism* ; Genetic Engineering/methods* ; Promoter Regions, Genetic ; Metabolic Engineering/methods* ; Fungal Proteins/genetics*

Energy metabolism regulation plays a pivotal role in metabolic engineering. It mainly achieves the balance of material and energy metabolism or maximizes the utilization of materials and energy by regulating the supply intensity and mode of ATP and reducing electron carriers in cells. On the one hand, the production efficiency can be increased by changing the distribution of material metabolic flow. On the other hand, the thermodynamic parameters of enzyme-catalyzed reactions can be altered to affect the reaction balance, and thus the production costs are reduced. Therefore, energy metabolism regulation is expected to become a favorable tool for the modification of microbial cell factories, thereby increasing the production of target metabolites and reducing production costs. This article introduces the commonly used energy metabolism regulation methods and their effects on cell factories, aiming to provide a reference for the efficient construction of microbial cell factories.
Energy Metabolism/physiology* ; Metabolic Engineering/methods* ; Adenosine Triphosphate/metabolism* ; Industrial Microbiology/methods*

The ability of cells to sense and adapt to metabolic changes and environmental variations is essential for their functions. Recent advances in synthetic biology have uncovered increasing mechanisms through which cells detect changes in metabolism and environmental conditions, leading to broader applications. However, a systematic review on the regulation of cellular metabolism and environmental adaption is currently lacking. This article presents a comprehensive overview of this field from three perspectives. First, it introduces key transmembrane and sensor proteins involved in the cellular perception of metabolic and environmental changes. Next, it summarizes the adaptive regulation mechanisms that natural cells employ when confronted with intracellular and extracellular metabolic changes. Finally, the review explores the application scenarios based on cellular adaptive regulation in three aspects: dynamic control, rational metabolic engineering, and adaptive evolution and makes an outlook on the future development directions in this field. This review not only provides a comprehensive perspective on the mechanisms by which cells sense metabolic and environmental variations, but also lays a theoretical foundation for further innovations in the field of synthetic biology. With the continuous advancement of future technologies, a deeper understanding of cellular adaptive regulation mechanisms holds great potential to drive the development and application of novel biomanufacturing platforms.
Adaptation, Physiological ; Synthetic Biology ; Metabolic Engineering/methods* ; Environment ; Bacteria/genetics*

Echinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin. It is a secondary metabolite of Aspergillus nidulans, and its titer in fermentation is significantly affected by the ECB synthesis pathway and cell morphology. In this study, the key genes related to the transcription activation, hydroxylation, and cell morphology during ECB biosynthesis were investigated to increase the fermentation titer of ECB and to change the cell morphology of Aspergillus nidulans to reduce the viscosity of the fermentation broth. The results indicated that after overexpression of ecdB and ecdK, the ECB titer increased by 25.8% and 23.7%, respectively, compared with that of the wild-type strain, reaching (2 030.5±99.2) mg/L and (1 996.4±151.4) mg/L. However, the deletion of fksA associated with cell wall synthesis resulted in damage to the cell wall, affecting strain growth and product synthesis. The engineered strain overexpressing ecdB was fermented in a 50-L bioreactor, in which the ECB titer reached 2 234.5 mg/L. The findings laid a research foundation for the subsequent metabolic engineering of this strain.
Fermentation ; Aspergillus nidulans/genetics* ; Echinocandins/genetics* ; Bioreactors/microbiology* ; Fungal Proteins/biosynthesis* ; Metabolic Engineering

Actinomycetes are important producers of high-value natural products, and the engineering of actinomycetes to enhance the biosynthesis of target natural products has long been a hot research topic in the scientific community. However, non-rational engineering methods suffer from low beneficial mutation rates, which limit the efficiency of mutant screening. The integration of high-throughput screening (HTS) technologies can effectively enhance the screening efficiency of elite mutants and significantly shorten the cycle of actinomycete strain engineering. This review comprehensively discusses various HTS technologies suitable for the engineering of actinomycete strains and compares them in terms of application scenarios, advantages, and disadvantages. HTS technologies include microplate-based screening, antimicrobial activity screening, antibiotic resistance screening, fluorescence-activated cell sorting (FACS), and fluorescence-activated droplet sorting (FADS). Additionally, this review summarizes the applications of these technologies in assisting actinomycete strain engineering and enhancing the yields of target compounds. The development and application of HTS technologies have not only facilitated the exploration of natural product resources in actinomycetes but also provided strong support for the rapid and efficient construction of high-performance engineered actinomycete strains.
Actinobacteria/metabolism* ; High-Throughput Screening Assays/methods* ; Genetic Engineering/methods* ; Biological Products/metabolism* ; Flow Cytometry ; Metabolic Engineering/methods*