Construction Of Cell Factories Research Articles

Advances on the microbial synthesis of plant-derived diterpenoids

Natural plant-derived diterpenoids are a class of compounds with diverse structures and functions. These compounds are widely used in pharmaceuticals, cosmetics and food additives industries because of their pharmacological properties such as anticancer, anti-inflammatory and antibacterial activities. In recent years, with the gradual discovery of functional genes in the biosynthetic pathway of plant-derived diterpenoids and the development of synthetic biotechnology, great efforts have been made to construct a variety of diterpenoid microbial cell factories through metabolic engineering and synthetic biology, resulting in gram-level production of many compounds. This article summarizes the construction of plant-derived diterpenoid microbial cell factories through synthetic biotechnology, followed by introducing the metabolic engineering strategies applied to improve plant-derived diterpenoids production, with the aim to provide a reference for the construction of high-yield plant-derived diterpenoid microbial cell factories and the industrial production of diterpenoids.

Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Application of Adaptive Laboratory Evolution in Lipid and Terpenoid Production in Yeast and Microalgae.

Due to the complexity of metabolic and regulatory networks in microorganisms, it is difficult to obtain robust phenotypes through artificial rational design and genetic perturbation. Adaptive laboratory evolution (ALE) engineering plays an important role in the construction of stable microbial cell factories by simulating the natural evolution process and rapidly obtaining strains with stable traits through screening. This review summarizes the application of ALE technology in microbial breeding, describes the commonly used methods for ALE, and highlights the important applications of ALE technology in the production of lipids and terpenoids in yeast and microalgae. Overall, ALE technology provides a powerful tool for the construction of microbial cell factories, and it has been widely used in improving the level of target product synthesis, expanding the range of substrate utilization, and enhancing the tolerance of chassis cells. In addition, in order to improve the production of target compounds, ALE also employs environmental or nutritional stress strategies corresponding to the characteristics of different terpenoids, lipids, and strains.

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone.

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

Improved protein production in yeast using cell engineering with genes related to a key factor in the unfolded protein response

The yeast Saccharomyces cerevisiae is a widely used cell factory for protein production. Increasing the protein production capacity of a yeast strain may be beneficial for obtaining recombinant proteins as a product or exerting its competence in consolidated bioprocessing. However, heterologous protein expression usually imposes stress on cells. Improving the cell's ability to cope with stress enhances protein yield. HAC1 is a key transcription factor in the unfolded protein response (UPR). In this study, several genes related to the UPR signal pathway, including unfolded protein sensing, HAC1 mRNA splicing, mRNA ligation, mRNA decay, translation, and Hac1p degradation, were selected as targets to engineer yeast strains. The final engineered strain produced α-amylase 3.3-fold, and human serum albumin 15.3-fold, greater than that of the control strain. Key regulation and metabolic network changes in the engineered strains were identified by transcriptome analysis and physiological characterizations. This study demonstrated that cell engineering with genes relevant to the key node HAC1 in UPR increased protein secretion substantially. The verified genetic modifications of this study provide useful targets in the construction of yeast cell factories for efficient protein production.

Microbial synthesis of glycosaminoglycans and their oligosaccharides.

Compared with chemical synthesis and tissue extraction methods, microbial synthesis of glycosaminoglycans (GAGs) is attractive because of the advantages of eco-friendly processes, production safety, and sustainable development. However, boosting the efficiency of microbial cell factories, precisely regulating GAG molecular weights, and rationally controlling the sulfation degree of GAGs remain challenging. To address these issues, various strategies, including genetic, enzymatic, metabolic, and fermentation engineering, have been developed. In this review, we summarize the recent progress in the construction of efficient GAG-producing microbial cell factories, regulation of the molecular weight of GAGs, and modification of GAG chains. Moreover, future studies, remaining challenges, and potential solutions in this field are discussed.

Adaptive evolution of microorganisms based on industrial environmental perturbations

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.

Establishment, optimization, and application of genetic technology in Aspergillus spp.

Aspergillus is widely distributed in nature and occupies a crucial ecological niche, which has complex and diverse metabolic pathways and can produce a variety of metabolites. With the deepening of genomics exploration, more Aspergillus genomic informations have been elucidated, which not only help us understand the basic mechanism of various life activities, but also further realize the ideal functional transformation. Available genetic engineering tools include homologous recombinant systems, specific nuclease based systems, and RNA techniques, combined with transformation methods, and screening based on selective labeling. Precise editing of target genes can not only prevent and control the production of mycotoxin pollutants, but also realize the construction of economical and efficient fungal cell factories. This paper reviewed the establishment and optimization process of genome technologies, hoping to provide the theoretical basis of experiments, and summarized the recent progress and application in genetic technology, analyzes the challenges and the possibility of future development with regard to Aspergillus.

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo.

In spite of the development of genome-editing tools using CRISPR-Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5'-phosphorylated (5'-P) or 5'- hydroxylated (5'-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn2+ or Mg2+ and displayed no obvious preference for the 5'-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.

Production of 3-Hydroxypropionic Acid from Renewable Substrates by Metabolically Engineered Microorganisms: A Review

3-Hydroxypropionic acid (3-HP) is a platform chemical with a wide range of existing and potential applications, including the production of poly(3-hydroxypropionate) (P-3HP), a biodegradable plastic. The microbial synthesis of 3-HP has attracted significant attention in recent years due to its green and sustainable properties. In this paper, we provide an overview of the microbial synthesis of 3-HP from four major aspects, including the main 3-HP biosynthesis pathways and chassis strains used for the construction of microbial cell factories, the major carbon sources used for 3-HP production, and fermentation processes. Recent advances in the biosynthesis of 3-HP and related metabolic engineering strategies are also summarized. Finally, this article provides insights into the future direction of 3-HP biosynthesis.

Production of 11-Oxo-β-Amyrin in Saccharomyces cerevisiae at High Efficiency by Fine-Tuning the Expression Ratio of CYP450:CPR

The production of glycyrrhetinic acid (GA) and 11-oxo-β-amyrin, the major bioactive components in liquorice, was typically inhibited by P450 oxidation in Saccharomyces cerevisiae. This study focused on optimizing CYP88D6 oxidation by balancing its expression with cytochrome P450 oxidoreductase (CPR) for the efficient production of 11-oxo-β-amyrin in yeast. Results indicated that a high CPR:CYP88D6 expression ratio could decrease both 11-oxo-β-amyrin concentration and turnover ratio of β-amyrin to 11-oxo-β-amyrin, whereas a high CYP88D6:CPR expression ratio is beneficial for improving the catalytic activity of CYP88D6 and 11-oxo-β-amyrin production. Under such a scenario, 91.2% of β-amyrin was converted into 11-oxo-β-amyrin in the resulting S. cerevisiae Y321, and 11-oxo-β-amyrin production was further improved to 810.6 mg/L in fed-batch fermentation. Our study provides new insights into the expression of cytochrome P450 and CPR in maximizing the catalytic activity of P450s, which could guide the construction of cell factories in producing natural products.

Automating the design-build-test-learn cycle towards next-generation bacterial cell factories

Automation is playing an increasingly significant role in synthetic biology. Groundbreaking technologies, developed over the past 20 years, have enormously accelerated the construction of efficient microbial cell factories. Integrating state-of-the-art tools (e.g. for genome engineering and analytical techniques) into the design-build-test-learn cycle (DBTLc) will shift the metabolic engineering paradigm from an almost artisanal labor towards a fully automated workflow. Here, we provide a perspective on how a fully automated DBTLc could be harnessed to construct the next-generation bacterial cell factories in a fast, high-throughput fashion. Innovative toolsets and approaches that pushed the boundaries in each segment of the cycle are reviewed to this end. We also present the most recent efforts on automation of the DBTLc, which heralds a fully autonomous pipeline for synthetic biology in the near future.

Dual-plasmid interactions stimulate the accumulation of valencene in Saccharomyces cerevisiae

Plasmids are one of the most commonly used basic tools in the construction of microbial cell factories, the use of which individually or in pairs play an important role in the expression of exogenous gene modules. However, little attention has been paid to the interactions of plasmid-plasmid and plasmid-host in the widespread use of the double plasmid system. In this study, we demonstrated that dual-plasmid interactions facilitated to cell growth and product accumulation in Saccharomyces cerevisiae. The strain containing both the expression plasmid pEV (a plasmid carrying the gene encoding valencene synthase) and the assistant plasmid pI (an empty plasmid expressing no extra gene) showed a significant improvement in relative growth rate, biomass and valencene production compared to the strain containing only the pEV plasmid. The transcriptional level analysis revealed an up-regulated expression of specific gene on the expression plasmid pEV stimulated by the assistant plasmid pI in the dual-plasmid interactions. Further investigations demonstrated the essential roles of the promoters of the expression plasmid pEV and the CEN/ARS element of the assistant plasmid pI in the dual-plasmid interactions. Combined with the results of predicted nucleosome occupancy, a response model of interaction based on the key T(n)C and CEN/ARS element was established. Moreover, the transformation order of the two plasmids significantly affected the response effect, implying the dominance of plasmid pI in the dual-plasmid interactions. Our finding first demonstrated that dual plasmids regulate the gene expression through spatial interactions at DNA sequences level, which provides a new perspective for the development of microbial cell factories in future.

An efficient marker-free genome editing method for Aspergillus niger

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.

Biosynthesis and regulation of diterpenoids in medicinal plants.

Plant diterpenoids are widely distributed and abundant natural products with diverse structures and functions in nature, which have been commonly used in pharmaceutical, agricultural and industrial production. In recent years, plant diterpenoids have attracted increasing attention, including their biosynthetic pathways, transcriptional regulatory networks, and biological functions. Herein, the biosynthetic pathways of diterpenoids are summarized in a modular fashion. Further, the regulatory network between diterpene biosynthesis and environmental factors is reviewed. Insights into diterpene metabolism may drive elucidation of complex active diterpene pathways and serve as a knowledge repository for metabolic engineering and cell factory construction.

Efficient fatty acid synthesis from methanol in methylotrophic yeast

Methanol is an attractive C1 feedstock with high abundance and low cost in bio-manufacturing. However, the metabolic construction of cell factories to utilize methanol for chemicals production remains a challenge due to the toxic intermediates and complicated metabolic pathways. The group of Zhou rescued methylotrophic yeast from cell death and achieved high-level production of free fatty acids from methanol through a combination of adaptive laboratory evolution, rational metabolic engineering and multi-omics analysis.

High-yield ectoine production in engineered Corynebacterium glutamicum by fine metabolic regulation via plug-in repressor library

Ectoine is a high-value protective and stabilizing agent with different applications in biopharmaceuticals, biotechnology, and fine chemicals. Here, efficient production of ectoine in Corynebacterium glutamicum was achieved by combination of metabolic engineering and plug-in repressor library strategy. First, the ectBAC cluster from Pseudomonas stutzeri was introduced into strain K02, and the titer of the obtained strain was 2.12 g/L. Metabolic engineering was then performed for further optimization, including removal of competing pathways (pck and ldh knockout), deletion of glycolysis repressor (sugR knockout), and enhancement of precursor supply (overexpression of Ecasd and CglysCS301Y). Next, two repressor libraries were designed for targeted flux control to improve ectoine production. Finally, strain CB5L6 produced 45.52 g/L ectoine and had the highest yield in C. glutamicum. For the first time, plug-in repressor library was employed to engineer C. glutamicum for metabolites production, which will provide a guideline for the construction of microbial cell factories.

CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae.

The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved α-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were reintroduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation), and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. A total of 11 point mutations and seven gene deletions were found to effectively improve α-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication, and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion.

Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius.

Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

Microbial synthesis of long-chain α-alkenes from methanol by engineering Pichia pastoris

α-Alkenes (terminal alkenes) are important fuel and platform chemicals that are mainly produced from petroleum. Microbial synthesis might provide a sustainable approach for α-alkenes. In this work, we engineered the methylotrophic yeast Pichia pastoris to produce long-chain (C15:1, C17:1 and C17:2) α-alkenes via a decarboxylation of fatty acids. Combinatorial engineering, including enzyme selection, expression optimization and peroxisomal compartmentalization, enabled the production of 1.6 mg/L α-alkenes from sole methanol. This study represents the first case of α-alkene biosynthesis from methanol and also provides a reference for the construction of methanol microbial cell factories of other high-value chemicals.Graphical