Xylose is a major component of lignocellulose and the second most abundant sugar present in nature. Efficient utilization of xylose is required for the development of economically viable processes to produce biofuels and chemicals from biomass. However, there are still some bottlenecks in the bioconversion of xylose, including the fact that some microorganisms cannot assimilate xylose naturally and that the uptake and metabolism of xylose are inhibited by glucose, which is usually present with xylose in lignocellulose hydrolysate. To overcome these issues, numerous efforts have been made to discover, characterize, and engineer the transporters and enzymes involved in xylose utilization to relieve glucose inhibition and to develop recombinant microorganisms to produce fuels and chemicals from xylose. Here we describe a recent advancement focusing on xylose-utilizing pathways, biosynthesis of chemicals from xylose, and engineering strategies used to improve the conversion efficiency of xylose.

Geraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown. Recombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4 % of volatile geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, most of geraniol was eventually biotransformed into geranyl acetate by E. coli, thus decreasing geraniol production. For the first time, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing geranyl acetate to geraniol, and production of geraniol was successfully increased to 2.0 g/L under controlled fermentation conditions. An efficient geraniol production platform was established by overexpressing several key pathway proteins in engineered E. coli strain combined with a controlled fermentation system. About 2.0 g/L geraniol was obtained using our controllable aqueous-organic two-phase fermentation system, which is the highest yield to date. In addition, the interconversion between geraniol and geranyl acetate by E. coli was first elucidated. This study provided a new and promising strategy for geraniol biosynthesis, which laid a basis for large-scale industrial application.

The biotechnological production of isoprene and isopentenol has recently been studied. Isoprene, which is currently made mainly from petroleum, is an important platform chemical for synthesizing pesticides, medicines, oil additives, fragrances, and more and is especially important in the rubber production industry. Isopentenols, which have better combustion properties than well-known biofuels (ethanol), have recently received more attention. Supplies of petroleum, the conventional source of isoprene and isopentenols, are unsustainable, and chemical synthesis processes could cause serious environmental problems. As an alternative, the biosynthesis of isoprene and isopentenols in cell factories is more sustainable and environmentally friendly. With a number of advantages over other microorganisms, Escherichia coli is considered to be a powerful workhorse organism for producing these compounds. This review will highlight the recent advances in metabolic engineering for isoprene and isopentenol production, especially using E. coli cell factories.

Acetyl-CoA-derived chemicals are suitable for multiple applications in many industries. The bio-production of these chemicals has become imperative owing to the economic and environmental problems. However, acetate overflow is the major drawback for acetyl-CoA-derived chemicals production. Approaches for overcoming acetate overflow may be beneficial for the production of acetyl-CoA-derived chemicals. In this study, a transcriptional regulator iclR was knocked out in E.coli BL21(DE3) to overcome acetate overflow and improve the chemicals production. Two important acetyl-CoA-derived chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) were used to evaluate it. It is revealed that knockout of iclR significantly increased expressions of aceBAK operon. The cell yields and glucose utilization efficiencies were higher than those of control strains. The acetate concentrations were decreased by more than 50% and the productions of PG and 3HP were increased more than twice in iclR mutants. The effects of iclR knockout on cell physiology, cell metabolism and production of acetyl-CoA-derived chemicals were similar to those of arcA knockout in our previous study. However, the arcA-iclR double mutants couldn’t gain higher productions of PG and 3HP. The mechanisms are unclear and needed to be resolved in future. Knockout of iclR significantly increased gene expression of aceBAK operon and concomitantly activated glyoxylate pathway. This genetic modification may be a good way to overcome acetate overflow, and improve the production of a wide range of acetyl-CoA-derived chemicals.

As an essential platform chemical mostly used for rubber synthesis, isoprene is produced in industry through chemical methods, derived from petroleum. As an alternative, bio-production of isoprene has attracted much attention in recent years. Previous researches were mostly focused on key enzymes to improve isoprene production. In this research, besides screening of key enzymes, we also paid attention to expression intensity of non-key enzymes. Firstly, screening of key enzymes, IDI, MK and IspS, from other organisms and then RBS optimization of the key enzymes were carried out. The strain utilized IDIsa was firstly detected to produce more isoprene than other IDIs. IDIsa expression was improved after RBS modification, leading to 1610-fold increase of isoprene production. Secondly, RBS sequence optimization was performed to reduce translation initiation rate value of non-key enzymes, ERG19 and MvaE. Decreased ERG19 and MvaE expression and increased isoprene production were detected. The final strain showed 2.6-fold increase in isoprene production relative to the original strain. Furthermore, for the first time, increased key enzyme expression and decreased non-key enzyme expression after RBS sequence optimization were obviously detected through SDS-PAGE analysis. This study prove that desired enzyme expression and increased isoprene production were obtained after RBS sequence optimization. RBS optimization of genes could be a powerful strategy for metabolic engineering of strain. Moreover, to increase the production of engineered strain, attention should not only be focused on the key enzymes, but also on the non-key enzymes.

Glycolate is a valuable chemical with extensive applications in many different fields. The traditional methods to synthesize glycolate are quite expensive and toxic. So, the biotechnological production of glycolate from sustainable feedstocks is of interest for its potential economic and environmental advantages. d-Xylose is the second most abundant sugar in nature and accounts for 18–30% of sugar in lignocellulose. New routes for the conversion of xylose to glycolate were explored. Overexpression of aceA and ghrA and deletion of aceB in Escherichia coli were examined for glycolate production from xylose, but the conversion was initially ineffective. Then, a new route for glycolate production was established in E. coli by introducing NAD+-dependent xylose dehydrogenase (xdh) and xylonolactonase (xylC) from Caulobacter crescentus. The constructed engineered strain Q2562 produced 28.82 ± 0.56 g/L glycolate from xylose with 0.60 ± 0.01 g/L/h productivity and 0.38 ± 0.07 g/g xylose yield. However, 27.18 ± 2.13 g/L acetate was accumulated after fermentation. Deletions of iclR and ackA were used to overcome the acetate excretion. An ackA knockout resulted in about 66% decrease in acetate formation. The final engineered strain Q2742 produced 43.60 ± 1.22 g/L glycolate, with 0.91 ± 0.02 g/L/h productivity and 0.46 ± 0.03 g/g xylose yield. A new route for glycolate production from xylose was established, and an engineered strain Q2742 was constructed from this new explored pathway. The engineering strain showed the highest reported productivity of glycolate to date. This research opened up a new prospect for bio-refinery of xylose and an alternative choice for industrial production of glycolate.