xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari

Abstract

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (Y = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.

Investigating Nutrient Limitation Role on Improvement of Growth and Poly(3-Hydroxybutyrate) Accumulation by Burkholderia sacchari LMG 19450 From Xylose as the Sole Carbon Source

“…In a different study, when cultivated under lower phosphorus concentration, B. sacchari growth rate was 0.15 1/h (Guamán et al, 2018), lower than the herein obtained under higher phosphorus concentrations (B101-X1 and -X3), which suggests that phosphorus concentration was limiting growth.…”

“…Both media are detailed in Table 1. Xylose was sterilized separately and aseptically added to the culture media; the final concentration used was 15-20 g/L, which was sufficient for cell growth until the start of P(3HB) accumulation phase considering previously obtained yield data (Guamán et al, 2018). FIGURE 2 | PHA (g/L, left y-axis) and content (%, right y-axis) obtained for each fed-batch cultivation.…”

Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineeredE. coli

“…XylA) Xylose isomerase quantificationXylose isomerase activities from cell extracts were quantified with a D-xylose reductase coupled enzyme assay, similar to methods previously described, and following a decrease in absorbance of NADPH at 340nm 56,57. Cultures were grown in shake flasks in SM10++ meda and harvested in mid exponential phase, washed and resuspended in SM 10 No phosphate media.After 16 hours of phosphate depletion, cells were pelleted by 10 minutes of centrifugation (4122 RCF, 4 degrees C) and lysed with BugBuster protein extraction reagent (Millipore Sigma, Catalog #70584) according to the manufacturer's protocol.…”

Engineering xylose metabolism for production of polyhydroxybutyrate in the non-model bacterium Burkholderia sacchari

“…The genes in the present study were selected using data from a recent analysis of B. sacchari genome from our group [ 30 ]. Minimum Tm of 60 °C and 18 bp hybridization to target was used as a standard for primer design.…”