Khusnutdinova, A. | Search bioRxiv

bioRxiv2023

One-pot chemo-enzymatic synthesis and one-step recovery of homogeneous long-chain polyphosphates from microalgal biomass

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/553819v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1fabe18org.highwire.dtl.DTLVardef@d65a45org.highwire.dtl.DTLVardef@c09593org.highwire.dtl.DTLVardef@19c1785_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG Phosphate, an essential component of life, fertilizers, and detergents, is a finite resource that could be depleted within 70 years, while improper phosphate waste disposal in aquatic environments results in eutrophication. Despite some chemical-based methods, biological phosphorus removal using polyphosphate-accumulating organisms, such as microalgae, serves as a sustainable alternative to reclaim phosphate from wastewater. Polyphosphates have profound biological functions and biomedical applications, serving as energy stock, coagulation factors, and antiviral agents depending on their length, showing inherent value in polyphosphate recovery. Here, we leveraged the power of thermodynamic coupling and phase transitions to establish a one-pot, two-step multi-enzyme cascade to convert polydisperse polyphosphate in microalgae biomass into high-molecular-weight insoluble long-chain polyphosphates, allowing for one-step purification. We then optimzed a thermo-digestion approach to transform the 1,300-mers into shorter polyphosphates. Altogether, the processes established here enable the establishment of a sustainable P bioeconomy platform to refine microalgal biomass for biotechnological uses.

synthetic biology↗

bioRxiv2018

Flux balance analysis predicts NADP phosphatase and NADH kinase are critical to balancing redox during xylose fermentation in Scheffersomyces stipitis

Xylose is the second most abundant sugar in lignocellulose and can be used as a feedstock for next-generation biofuels by industry. Saccharomyces cerevisiae, one of the main workhorses in biotechnology, is unable to metabolize xylose natively but has been engineered to ferment xylose to ethanol with the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Scheffersoymces stipitis. In the scientific literature, the yield and volumetric productivity of xylose fermentation to ethanol in engineered S. cerevisiae still lags S. stipitis, despite expressing of the same XR-XDH genes. These contrasting phenotypes can be due to differences in S. cerevisiaes redox metabolism that hinders xylose fermentation, differences in S. stipitis redox metabolism that promotes xylose fermentation, or both. To help elucidate how S. stipitis ferments xylose, we used flux balance analysis to test various redox balancing mechanisms, reviewed published omics datasets, and studied the phylogeny of key genes in xylose fermentation. In vivo and in silico xylose fermentation cannot be reconciled without NADP phosphatase (NADPase) and NADH kinase. We identified eight candidate genes for NADPase. PHO3.2 was the sole candidate showing evidence of expression during xylose fermentation. Pho3.2p and Pho3p, a recent paralog, were purified and characterized for their substrate preferences. Only Pho3.2p was found to have NADPase activity. Both NADPase and NAD(P)H-dependent XR emerged from recent duplications in a common ancestor of Scheffersoymces and Spathaspora to enable efficient xylose fermentation to ethanol. This study demonstrates the advantages of using metabolic simulations, omics data, bioinformatics, and enzymology to reverse engineer metabolism.

microbiology↗