Microbial production of fuels via bioprocesses has emerged as an attractive alternative to the traditional production of fuels. Wiparat Siripong of the National Center for Genetic Engineering and Biotechnology in Thailand engineered the yeast Pichia pastoris to produce isobutanol from glucose and glycerol. The team diverted the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production.

The engineered strain overexpressing the keto-acid degradation pathway produced 284 mg/L of isobutanol when fed with 2-ketoisovalerate. To improve the production of isobutanol, the team overexpressed a part of the amino acid l-valine biosynthetic pathway in the engineered strain. This led to a strain capable of producing 0.89 g/L of isobutanol. Fine-tuning the expression of bottleneck enzymes further improved the production titer of isobutanol by 43 times compared to the original strain.

This work will provide a route to establish P. pastoris as a versatile production platform for fuels and chemicals.

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols. However, the produced alcohol is toxic to Synechocystis sp., hindering industrial applications. Hence, researchers want to engineer organisms with strong alcohol tolerance. Takuya Matsusako of Osaka University in Japan aimed to increase alcohol tolerance genes in Synechocystis sp. via adaptive laboratory evolution.

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains were capable of growing on medium with 5 g/L isobutanol, on which the parental strain could not grow. Analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of several genes including slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD.

The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols. And with the introduction of an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Second-generation biofuels help decrease dependency on fossil fuels. To make biomass more suitable for biorefinery use, researchers need to better understand plant cell wall synthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for biofuel production. Aude Aznar and Camille Chalvin of Lawrence Berkeley National Laboratory have identified genes involved in the synthesis of pectic galactan, including the GALS1 and URGT1.

Their team aimed to engineer plants with increased pectic galactan in stems. To do this, they used plants that were already engineered to have low xylan content, then further engineered them to overexpress GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Finally, the high galactan and low xylan traits were again stacked with the low lignin trait, which was achieved by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells.

These results show that increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner, leading to improved properties in terms of biofuel production, without any negative growth effects.