The cyanobacterium Synechocystis sp. PCC 6803 has gained scientific interest for its potential to use solar energy and atmospheric CO2 for the production of high-value chemicals like pharmaceuticals, flavors, and fragrances. Forskolin is a diterpenoid found in the root cork of the plant Plectranthus barbatus and its biosynthetic pathway is initiated by two terpene synthases that convert geranylgeranyl diphosphate (GGDP) into the precursor 13-R-manoyl oxide (13-R-MO). Using the cyanobacterium Synechocystis sp. PCC 6803 as host, we expressed the two terpene synthases resulting in the synthesis of 0.83 mg/L 13-R-MO. Three different geranylgeranyl diphosphate synthases (GGDPSs) were selected for screening; a prokaryotic (Synechococcus sp. JA-3-3Ab (Sj)), a yeast (Saccharomyces cerevisiae (Sc)), and a plant (P. barbatus (Pb)) derived GGDPS. Strains containing the prokaryotic Sj- or the yeast ScGGDPS consistently yielded more 13-R-MO than the base strain. By overexpression of 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) positioned at the entry of the 2-C-methyl-d-erythritol 4-phosphate pathway (MEP) together with the prokaryotic SjGGDPS, the 13-R-MO titer was increased 11-fold to reach 9.7 mg/L by boosting the synthesis of GGDP, the direct substrate for the diterpenoid synthases. We further show that application of a n-dodecane overlay to remove 13-R-MO from the culture medium provided a 2–3 fold increase of the 13-R-MO in a separate cultivation system.
Green and efficient biocatalytic technology has become a complementary or alternative means of organic synthesis. Chemicals with two functional groups, such as α,ω-dicarboxylic acids, ω-amino fatty acids and ω-hydroxy fatty acids, are widely used in the synthesis of polymers such as polyesters and polyamides. In recent years, the production of biodegradable materials using renewable and abundant vegetable oils as green raw materials has attracted increasing attention, receiving an additional impetus from synthetic biology. This paper presents the recent research progress in the production of bifunctional chemicals with medium chain lengths of C8–C12 using multi-enzyme cascades. Recent studies have developed multilevel optimization strategies to improve the efficiency, economics, and sustainability of multi-enzyme cascades. Cofactor regeneration strategies were developed to avoid large additions of expensive coenzymes. Protein engineering strategies were applied to improve enzyme stability and catalytic performance. In addition, blocking the β-oxidation pathway, improving the efficiency of substrate transport across membranes and increasing cellular robustness are effective optimization strategies for whole-cell catalytic systems. In addition, we discuss the development prospects of producing high value-added fine chemicals from vegetable oils using one-pot multi-enzyme reaction systems.
Terpenoids are a large class of secondary metabolites known for their remarkable diverse biological activities, making them widely utilized in the pharmaceutical, food, cosmetic, biofuel and agricultural fields. However, the current production of terpenoids heavily relies on plant extraction and chemical synthesis, which brings about concerns regarding infield, environmental and ecological issues. With the advancements in metabolic engineering and emerging synthetic biology tools, it is now possible to sustainably produce these high value-added terpenoids using microbial chassis. Among them, yeast has emerged as a promising candidate for the heterologous biosynthesis of terpenoids due to its inherent advantages, including robustness, safety, and the availability of sufficient precursor. This review focuses on the diverse strategies employed to enable terpenoids production in yeasts. These strategies encompass metabolic engineering approaches to optimize the mevalonate pathway, protein engineering techniques to improve terpenoid biosynthesis, the applications of organelles compartmentalization, high throughput screening and global approaches for the development of efficient cell factories. Furthermore, this review discusses the future prospects and challenges associated with yeast-based terpenoid production, while also emphasizing guidelines for future studies in this field.
Uroporphyrin (UP) is a porphyrin compound with medical applications and a key precursor for heme biosynthesis. However, there is no biosynthetic strategy for UP production. In this study, we present a novel bioprocess for enhanced production of UP in engineered Escherichia coli. We first implemented the Shemin/C4 pathway heterologously in an E. coli strain with an enlarged intracellular pool of succinyl-CoA. Using a plasmid with the trc promoter regulating the expression of a synthesized gene operon, the effects of key pathway genes, including hemA, hemB, hemC, and hemD, on UP biosynthesis were characterized. By cultivating the resulting engineered E. coli strains in a batch bioreactor with 30 g/L glycerol under aerobic conditions, up to 901.9 mg/L UP was produced. Most of the synthesized UP was extracellularly secreted with a high purity more than 80 wt%, facilitating its downstream purification. The study paves the way for large-scale bio-based production of UP using synthetic biology and metabolic engineering strategies.
Photofermentative hydrogen production with non-sulfur purple bacteria like Cereibacter sphaeroides (formerly Rhodobacter sphaeroides) is a promising and sustainable process to convert organic waste into the energy carrier hydrogen gas. However, this conversion is inhibited by elevated organic nitrogen concentrations in the substrate, which limits its applicability to nitrogen-poor organic waste. We present genomic and transcriptomic insights into a substrain of Cereibacter sphaeroides strain 2.4.1 that shows unexpected high levels of photofermentative hydrogen evolution when fed with glutamate. Genome sequencing revealed 222 single nucleotide variances (SNVs) between the reference genome of C. sphaeroides strain 2.4.1 and the analyzed substrain H2. These affect 61 protein coding genes. A leucine-proline exchange is present in the σ54 factor (rpoN2 gene), a global hydrogen and nitrogen metabolism regulator. We propose a model how this mutation alters DNA-binding properties that explain the unexpected organic nitrogen tolerance of hydrogen production. Transcriptomic analyses under varying glutamate concentrations support this finding. Thus, we present the first thorough genomic and transcriptomic analysis of a Cereibacter strain that shows promising metabolic characteristics for biotechnological hydrogen gas production from organic waste. These results suggest a potential target for strain optimization. Possibly, our key finding can be transferred to other hydrogen producing microorganisms.
Pattern formation is a fundamental process in biological development, enabling the transformation of initially uniform or random states into spatially ordered structures. A comprehensive understanding of the formation and function of these patterns is crucial for unraveling the underlying principles of biological design and engineering. In recent years, synthetic biology has emerged as a powerful discipline for investigating and manipulating pattern formation in biological systems, involving the design and construction of novel biological components, circuits, and networks with specific functionalities. The integration of computational simulations (in silico) and experimental techniques (wet lab) in synthetic biology has significantly advanced our knowledge of pattern formation and its implications in biological design and engineering. This review provides an overview of the computational simulations employed in studying pattern formation and introduces the representative and cutting-edge experimental methods utilized in wet labs.
Serine integrases are emerging as one of the powerful tools for synthetic biology. They have been widely developed across genome engineering, biological part construction, genetic circuit design, and in vitro DNA assembly. However, the strategy of in vivo DNA assembly by serine integrases has not yet been reported. To address this opportunity, here we developed a serine integrase-based in vivo DNA (plasmid) assembly approach. First, we demonstrated that the engineered “Acceptor” plasmids could be assembled with diverse “Donor” plasmids by serine integrases (Bxb1 and phiC31) in Escherichia coli (E. coli). Then, by programming the “Donor” plasmids and the host E. coli cells, we established an assembly cascade procedure and finally constructed plasmids that could constitutively express three different fluorescent proteins. Moreover, we used this approach to assemble different chromoprotein genes and generated colored E. coli cells. We anticipate that this approach will enrich the serine integrase-based biotechnology toolbox, and accelerate multiple plasmid assembly for synthetic biology with broad applications.
Fast, flexible and non-randomized modification, production and screening of proteins in fully automated system are of high interest in biological research and applications. The conventional methods for protein engineering and screening, especially for mutations of multiple residues. are time consuming and often unreliable. We demonstrate here a new, fast and flexible protein production and screening method which combines linear expression template (LET) based cell free protein synthesis (CFPS) with specific screening methods. This approach is demonstrated using green fluorescence protein, phosphoserine aminotransferase (serC) and aspartokinase III (AKIII) as model systems. The results show that mutants with changes in different protein properties upon multiple point mutations can be produced and screened within 6 to 15 h. This method can be used further to generate mutants of enzymes and multi-enzyme complexes and be implemented within the workflow of a feedback-guided protein optimization and screening system.
Five-carbon (C5) and six-carbon (C6) chemicals are essential components in the manufacturing of a variety of pharmaceuticals, fuels, polymers, and other materials. However, the predominant reliance on chemical synthesis methods and unsustainable feedstock sources has placed significant strain on Earth’s finite fossil resources and the environment. To address this challenge and promote sustainability, significant efforts have been undertaken to re-program microorganisms through metabolic engineering and synthetic biology approaches allowing for bio-based manufacturing of these compounds. This review provides a comprehensive overview of the advancements in microbial production of commercially significant non-natural C5 chemicals, including 1-pentanol, 1,5-pentanediol, cadaverine, δ-valerolactam, glutaric acid, glutaconic acid, and 5-hydroxyvaleric acid, as well as C6 chemicals, including cis, cis-muconic acid, adipic acid, 1,6-hexamethylenediamine, 6-aminocaproic acid, β-methyl-δ-valerolactone, 1-hexanol, ε-caprolactone, 6-hydroxyhexanoic acid, and 1,6-hexanediol.
