Pharmaceutically important polyketides such as avermectin are mainly produced as secondary metabolites during the stationary phase of growth of Streptomyces species in fermenters. The source of intracellular metabolites that are funneled into polyketide biosynthesis has proven elusive. We applied multi-omics to reveal that intracellular triacylglycerols (TAGs), which accumulates in primary metabolism, are degraded during stationary phase. This process could channel carbon flux from both intracellular TAGs and extracellular substrates into polyketide biosynthesis. We devised a strategy named ‘dynamic degradation of TAG’ (ddTAG) to mobilize the TAG pool and increase polyketide biosynthesis. Using ddTAG we increased the titers of actinorhodin, jadomycin B, oxytetracycline and avermectin B1a in Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces rimosus and Streptomyces avermitilis. Application of ddTAG increased the titer of avermectin B1a by 50% to 9.31 g l−1 in a 180-m3 industrial-scale fermentation, which is the highest titer ever reported. Our strategy could improve polyketide titers for pharmaceutical production. Polyketide yields in Streptomyces are boosted by routing stored intracellular triacylglycerol into pathways that make industrially relevant products.

Harnessing the intracellular triacylglycerols for titer improvement of polyketides in Streptomyces.

Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey

Natural nonproteinogenic amino acids vastly outnumber the well-known 22 proteinogenic amino acids. Such amino acids are generated in specialized metabolic pathways. In these pathways, diverse biosynthetic transformations, ranging from isomerizations to the stereospecific functionalization of C-H bonds, are employed to generate structural diversity. The resulting nonproteinogenic amino acids can be integrated into more complex natural products. Here we review recently discovered biosynthetic routes to freestanding nonproteinogenic α-amino acids, with an emphasis on work reported between 2013 and mid-2019.

Biosynthetic Pathways to Nonproteinogenic α-Amino Acids.

Three dimensional sea-urchin-like PdAuCu nanocrystals/ferrocene-grafted-polylysine as an efficient probe to amplify the electrochemical signals for ultrasensitive immunoassay of carcinoembryonic antigen.

Yarrowia lipolytica is a promising platform for single cell oil production. It is well-known for its metabolism oriented toward utilization of hydrophobic substrates and accumulation of storage lipids. Multiple copies of DGA2 under constitutive promoter were introduced into the Q4 strain, a quadruple mutant deleted for the four acyltransferases (Δdga1, Δdga2, Δlro1, and Δare1) to improve lipid accumulation. The Q4-DGA2 x3 strain contains three copies of DGA2. Further increase in accumulation was accomplished by blocking the β-oxidation pathway through MFE1 gene deletion yielding Q4-Δmfe DGA2 x3. In order to use molasses as a substrate for single cell oil production, sucrose utilization was established by expressing the Saccharomyces cerevisiae SUC2 gene yielding Q4-SUC2 DGA2 x3 and Q4-Δmfe SUC2 DGA2 x3. During cultivation on sucrose medium with a carbon to nitrogen ratio of 80, both strains accumulated more than 40 % of lipids, which was a 2-fold increase in lipid storage. Q4-Δmfe SUC2 DGA2 x3 accumulated more lipids than Q4-SUC2 DGA2 x3 (49 vs. 43 %) but yielded less biomass (13.7 vs. 15 g/L). When grown on 8 % (v/v) molasses, both strains accumulated more than 30 % of lipids after 3 days, while biomass yield was higher in Q4-SUC2 DGA2 x3 (16.4 vs. 14.4 g/L). Further addition of molasses at 72 h resulted in higher biomass yield, 26.6 g/L for Q4-SUC2 DGA2 x3, without modification of lipid content. This work presents genetically modified strains of Y. lipolytica as suitable tools for direct conversion of industrial molasses into value added products based on single cell oils.

Single cell oil production on molasses by Yarrowia lipolytica strains overexpressing DGA2 in multicopy

Effects of oxygen-vectors on the synthesis of epsilon-poly-lysine and the metabolic characterization of Streptomyces albulus PD-1

Economical production of poly(ε-l-lysine) and poly(l-diaminopropionic acid) using cane molasses and hydrolysate of streptomyces cells by Streptomyces albulus PD-1.

Poly(e-l-lysine) (e-PL) producer strain Streptomyces albulus PD-1 secreted a novel polymeric substance into its culture broth along with e-PL. The polymeric substance was purified to homogeneity and identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance spectroscopy as well as other analytical techniques revealed that the substance was poly(l-diaminopropionic acid) (PDAP). PDAP is an l-α,β-diaminopropionic acid oligomer linking between amino and carboxylic acid functional groups. The molecular weight of PDAP ranged from 500 to 1500 Da, and no co-polymers composed of l-diaminopropionic acid and l-lysine were present in the culture broth. Compared with e-PL, PDAP exhibited stronger inhibitory activities against yeasts but weaker activities against bacteria. e-PL and PDAP co-production was also investigated. Both e-PL and PDAP were synthesized during the stationary phase of growth, and the final e-PL and PDAP concentration reached 21.7 and 4.8 g L-1, respectively, in fed-batch fermentation. Citric acid feeding resulted in a maximum e-PL concentration of 26.1 g L-1 and a decrease in the final concentration of PDAP to 3.8 g L-1. No studies on e-PL and PDAP co-production in Streptomyces albulus have been reported previously, and inhibition of by-products such as PDAP is potentially useful in e-PL production.

Poly( l -diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly(ε- l -lysine) by Streptomyces albulus PD-1

Streptomyces albulus PD-1 can co-produce antimicrobial homo-polymers poly(e-lysine) (e-PL) and poly(L-diaminopropionic acid) (PDAP). In this study, a novel feeding strategy of citric acid coupled with glucose-(NH4)2SO4 feeding was employed to S. albulus PD-1. When the pH of the culture broth dropped to 4.0, the feeding solution was added continuously to maintain the concentrations of glucose and citric acid at 10 and 4 g L(-1), respectively. As a result, the final concentration of e-PL increased from 21.7 to 29.7 g L(-1) and the final concentration of PDAP decreased from 4.8 to 3.2 g L(-1). Assays on intracellular nucleotide levels and key enzyme activities were performed to elucidate the underlying regulation mechanism. The addition of citric acid increased NADH/NAD(+) ratio and decreased intracellular ATP level; meanwhile, the activities of pyruvate kinase, citrate synthase and isocitrate dehydrogenase decreased while aspartate aminotransferase activity increased. Therefore, we deduced that citric acid feeding resulted in metabolic flux redistribution at the node of phosphoenolpyruvate; the metabolic pathway from phosphoenolpyruvate directed into tricarboxylic acid cycle was weakened and thus PDAP production was inhibited. On the other hand, the metabolic pathway from phosphoenolpyruvate directed into oxaloacetate and L-aspartate was enhanced, thereby improving e-PL production. This fermentation strategy may be potentially useful in e-PL production because it can effectively inhibit the formation of by-products, such as PDAP.

Jun Xia

Papers

Effects of oxygen-vectors on the synthesis of epsilon-poly-lysine and the metabolic characterization of Streptomyces albulus PD-1

Economical production of poly(ε-l-lysine) and poly(l-diaminopropionic acid) using cane molasses and hydrolysate of streptomyces cells by Streptomyces albulus PD-1.

Poly( l -diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly(ε- l -lysine) by Streptomyces albulus PD-1

The regulatory effect of citric acid on the co-production of poly(ε-lysine) and poly(l-diaminopropionic acid) in Streptomyces albulus PD-1

A novel agar diffusion assay for qualitative and quantitative estimation of ε-polylysine in fermentation broths and foods