The cytosolic aminotransferase VAS1 coordinates aromatic amino acid biosynthesis and metabolism.

The aromatic amino acids (AAAs) phenylalanine, tyrosine, and tryptophan are basic protein units and precursors of diverse specialized metabolites that are essential for plant growth. Despite their significance, the mechanisms that regulate AAA homeostasis remain elusive. Here, we identified a cytosolic aromatic aminotransferase, REVERSAL OF SAV3 PHENOTYPE 1 (VAS1), as a suppressor of arogenate dehydrogenase 2 (adh2) in Arabidopsis (Arabidopsis thaliana). Genetic and biochemical analyses determined that VAS1 uses AAAs as amino donors, leading to the formation of 3-carboxyphenylalanine and 3-carboxytyrosine. These pathways represent distinct routes for AAA metabolism that are unique to specific plant species. Furthermore, we show that VAS1 is responsible for cytosolic AAA biosynthesis, and its enzymatic activity can be inhibited by 3-carboxyphenylalanine. These findings provide valuable insights into the crucial role of VAS1 in producing 3-carboxy AAAs, notably via recycling of AAAs in the cytosol, which maintains AAA homeostasis and allows plants to effectively coordinate the complex metabolic and biosynthetic pathways of AAAs.

Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine.

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.

Applications of synthetic biology in medical and pharmaceutical fields.

Synthetic biology aims to design or assemble existing bioparts or bio-components for useful bioproperties. During the past decades, progresses have been made to build delicate biocircuits, standardized biological building blocks and to develop various genomic/metabolic engineering tools and approaches. Medical and pharmaceutical demands have also pushed the development of synthetic biology, including integration of heterologous pathways into designer cells to efficiently produce medical agents, enhanced yields of natural products in cell growth media to equal or higher than that of the extracts from plants or fungi, constructions of novel genetic circuits for tumor targeting, controllable releases of therapeutic agents in response to specific biomarkers to fight diseases such as diabetes and cancers. Besides, new strategies are developed to treat complex immune diseases, infectious diseases and metabolic disorders that are hard to cure via traditional approaches. In general, synthetic biology brings new capabilities to medical and pharmaceutical researches. This review summarizes the timeline of synthetic biology developments, the past and present of synthetic biology for microbial productions of pharmaceutics, engineered cells equipped with synthetic DNA circuits for diagnosis and therapies, live and auto-assemblied biomaterials for medical treatments, cell-free synthetic biology in medical and pharmaceutical fields, and DNA engineering approaches with potentials for biomedical applications.

PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene.

Food wastes can be hydrolyzed into soluble microbial substrates, contributing to sustainability. Halomonas spp.-based Next Generation Industrial Biotechnology (NGIB) allows open, unsterile fermentation, eliminating the need for sterilization to avoid the Maillard reaction that negatively affects cell growth. This is especially important for food waste hydrolysates, which have a high nutrient content but are unstable due to batch, sources, or storage conditions. These make them unsuitable for polyhydroxyalkanoate (PHA) production, which usually requires limitation on either nitrogen, phosphorous, or sulfur. In this study, H. bluephagenesis was constructed by overexpressing the PHA synthesis operon phaCABCn (cloned from Cupriavidus necator) controlled by the essential gene ompW (encoding outer membrane protein W) promoter and the constitutive porin promoter that are continuously expressed at high levels throughout the cell growth process, allowing poly(3-hydroxybutyrate) (PHB) production to proceed in nutrient-rich (also nitrogen-rich) food waste hydrolysates of various sources. The recombinant H. bluephagenesis termed WZY278 generated 22 g L-1 cell dry weight (CDW) containing 80 wt% PHB when cultured in food waste hydrolysates in shake flasks, and it was grown to 70 g L-1 CDW containing 80 wt% PHB in a 7-L bioreactor via fed-batch cultivation. Thus, unsterilizable food waste hydrolysates can become nutrient-rich substrates for PHB production by H. bluephagenesis able to be grown contamination-free under open conditions.

Engineered Halomonas spp. for production of l-Lysine and cadaverine.

Cadaverine, a derivative of l-lysine, has been used as a monomer for the synthesis of bio-based nylon-5,6. This study engineered Halomonas bluephagenesis TD1.0 by blocking the feedback inhibition, overexpressing the key l-lysine synthesis genes, strengthening the l-lysine export system and increasing the supply of oxaloacetate for production of l-lysine in the supernatant and PHB in the cells. Subsequently, cadaverine biosynthetic pathway was constructed in H. campaniensis LC-9 to improve the efficiency of de novo cadaverine biosynthesis which combines l-lysine producing H. bluephagenesis TDL8-68-259 and cadaverine producing H. campaniensis LC-9-ldcC-lysP. When H. campaniensis LC-9-ldcC-lysP was used as a whole cell catalysis for cadaverine production, the conversion efficiency of l-lysine to cadaverine reached 100% in the presence of 0.05% Triton X-100 for cell membrane permeability enhancement, resulting in 118 g L-1 cadaverine formed in the fermentor. Thus, Halomonas spp. have been successfully constructed for l-lysine and cadaverine production.

The roadmap of bioeconomy in China.

The bioeconomy drives the development of life science and biotechnology as a blueprint for the future development of human society, and offers a cross-cutting perspective on the societal transformation towards long-term sustainability and the transition away from the non-renewable economy. Moreover, the sustainable bioeconomy strategies are consistent with the United Nation's (UN) Sustainable Development Goals (SDG) and are becoming the centre of the achievement for SDG. The Chinese '14th Five-Year Plan for Bioeconomy Development' (2021-2025), including the development goals of China's bioeconomy containing biomedicine, agriculture, bio-manufacturing and bio-security as a strategic priority, is discussed. The plan offers three pathways to improve bioeconomy, including technological innovation, industrialisation and policy supports. Finally, it concludes China's first bioeconomy development plan as a success, suggesting the key role of industrial biotechnology in bioeconomy.

Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis.

Flavonoids are major secondary metabolites derived from the plant phenylpropanoid pathway that play important roles in plant development and also have benefits for human health. So-called MBW ternary complexes involving R2R3-MYB and basic helix-loop-helix (bHLH) transcription factors along with WD-repeat proteins have been reported to regulate expression of the biosynthetic genes in the flavonoid pathway. MYB4 and its closest homolog MYB7 have been suggested to function as repressors of phenylpropanoid metabolism. However, the detailed mechanism by which they act has not been fully elucidated. Here, we show that Arabidopsis thaliana MYB4 and its homologs MYB7 and MYB32 interact with the bHLH transcription factors TT8, GL3 and EGL3 and thereby interfere with the transcriptional activity of the MBW complexes. In addition, MYB4 can also inhibit flavonoid accumulation by repressing expression of the gene encoding Arogenate Dehydratase 6 (ADT6), which catalyzes the final step in the biosynthesis of phenylalanine, the precursor for flavonoid biosynthesis. MYB4 potentially represses not only the conventional ADT6 encoding the plastidial enzyme but also the alternative isoform encoding the cytosolic enzyme. We suggest that MYB4 plays dual roles in modulating the flavonoid biosynthetic pathway in Arabidopsis.

MYB20, MYB42, MYB43, and MYB85 Regulate Phenylalanine and Lignin Biosynthesis during Secondary Cell Wall Formation.

Lignin is a phenylpropanoid-derived polymer that functions as a major component of cell walls in plant vascular tissues. Biosynthesis of the aromatic amino acid Phe provides precursors for many secondary metabolites, including lignins and flavonoids. Here, we discovered that MYB transcription factors MYB20, MYB42, MYB43, and MYB85 are transcriptional regulators that directly activate lignin biosynthesis genes and Phe biosynthesis genes during secondary wall formation in Arabidopsis (Arabidopsis thaliana). Disruption of MYB20, MYB42, MYB43, and MYB85 resulted in growth development defects and substantial reductions in lignin biosynthesis. In addition, our data showed that these MYB proteins directly activated transcriptional repressors that specifically inhibit flavonoid biosynthesis, which competes with lignin biosynthesis for Phe precursors. Together, our results provide important insights into the molecular framework for the lignin biosynthesis pathway.