Huaiwei Liu

High yield production of D-xylonic acid from D-xylose using engineered Escherichia coli.

An engineered Escherichia coli was constructed to produce D-xylonic acid, one of the top 30 high-value chemicals identified by US Department of Energy. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by disrupting xylose isomerase (XI) and xylulose kinase (XK) genes. The native pathway for xylonic acid catabolism was also blocked by disrupting two genes both encoding xylonic acid dehydratase (yagE and yjhG). Through the introduction of a D-xylose dehydrogenase from Caulobacter crescentus, a D-xylonic acid producing E. coli was constructed. The recombinant E. coli produced up to 39.2 g L(-1) D-xylonic acid from 40 g L(-1) D-xylose in M9 minimal medium. The average productivity was as high as 1.09 g L(-1) h(-1) and no gluconic acid byproduct was produced. These results suggest that the engineered E. coli has a promising application for the industrial-scale production of D-xylonic acid.

Combination of Entner-Doudoroff pathway with MEP increases isoprene production in engineered Escherichia coli.

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.

Combining De Ley-Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from D-galactose.

Abstract An engineered Escherichia coli strain was developed for enhanced isoprene production using d-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley–Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from d-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct d-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze d-galactose conversion to d-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD–MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD–MEP pathway.

l-arabonate and d-galactonate production by expressing a versatile sugar dehydrogenase in metabolically engineered Escherichia coli

The production of L-arabonate and D-galactonate employing a versatile l-arabinose dehydrogenase (AraDH) from Azospirillum brasilense is presented. The promiscuity of AraDH is manifested by its appreciable activity towards L-arabinose and D-galactose as substrates, and NAD(+) and NADP(+) as cofactors. The AraDH was introduced into an engineered Escherichia coli with inactive L-arabinose or D-galactose metabolism, resulting in strains EMA2 and EWG4, respectively. EMA2 produced 43.9 g L(-1)L-arabonate with a productivity of 1.22 g L(-1)h(-1) and 99.1% (mol/mol) yield. After methanol precipitation, 92.6% of L-arabonate potassium salt was recovered with a purity of 88.8%. Meanwhile, EWG4 produced 24.0 g L(-1)D-galactonate, which is 36% higher than that of the strain carrying the specific d-galactose dehydrogenase. Overall results reveal that the versatility of AraDH to efficiently catalyze the formation of L-arabonate and D-galactonate could be a useful tool in advancing industrial viability for sugar acids production.