Biofuels and bio-based chemicals from lignocellulose: metabolic engineering strategies in strain development

Interest in developing a sustainable technology for fuels and chemicals has unleashed tremendous creativity in metabolic engineering for strain development over the last few years. This is driven by the exceptionally recalcitrant substrate, lignocellulose, and the necessity to keep the costs down for commodity products. Traditional methods of gene expression and evolutionary engineering are more effectively used with the help of synthetic biology and -omics techniques. Compared to the last biomass research peak during the 1980s oil crisis, a more diverse range of microorganisms are being engineered for a greater variety of products, reflecting the broad applicability and effectiveness of today’s gene technology. We review here several prominent and successful metabolic engineering strategies with emphasis on the following four areas: xylose catabolism, inhibitor tolerance, synthetic microbial consortium, and cellulosic oligomer assimilation.     

PLA nanovectors with encapsulated betulin: plant leaf extract-synthesized nanovectors are more efficacious than PVA-synthesized nanovectors

Objectives

Betulin (BT) is an abundant triterpene found predominantly in the bark of Himalayan birch. It is difficult to deliver it in vivo because of its low aqueous solubility. We have therefore developed novel formulations of BT for improving its solubility, bioavailability and therapeutic efficacy.

Results

Poly-d,l-lactide nanovectors (PLA NVs) were synthesized using poly(vinyl alcohol) and Lonicera japonica leaf extract (LE) as a stabiliser and named as PLA-1 NVs and PLA-2 NVs. PLA-1 NVs and PLA-2 NVs were used for the encapsulation of betulin (BT) and named as BT-En-1 and BT-En-2 NVs. The encapsulation efficiency of BT-En-1 and BT-En-2 NVs were 99.3 and 100 % respectively. Prepared nanoformulations were physically stable. An in vitro study revealed 45 % BT was released over 24 h. BT had a prolonged release from BT-En-2 NVs as compared to BT-En-1 NVs. BT-En-2 NVs had better anticancerous activity against SiHa cells than BT-En-1 NVs.

Conclusions

Developed BT-EN-2 NVs had better biocompatibility, excellent stability and enhanced release characteristics than BT-En-1 NVs.

     

A cold-adapted and glucose-stimulated type II α-glucosidase from a deep-sea bacterium <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. K8

Objectives

To express and characterize a putative α-glucosidase, Pagl, from Pseudoalteromonas sp. K8 obtained via genome mining approach.

Results

Pagl was expressed and purified to homogeneity, with a molecular mass of 60 kDa. It was optimally active at pH 8.5 and 30 °C, and showed cold-adapted activity. Pagl exhibited specific activity towards substrates with α-1,4-linkage, with the highest specific activity of 19.4 U/mg for maltose, followed by pNPαG and maltodextrins, suggesting that Pagl belongs to the type II α-glucosidase. Interestingly, the activity of Pagl is significantly enhanced (2.7 times) in the presence of 200 mM glucose.

Conclusion

The unique catalytic properties of Pagl make it an attractive candidate for several industrial applications.

     

Endoglucanase enzymatic modification of kraft pulp during recycling

Objective

To investigate the cellulose modification process on kraft pulp during recycling by mono-endoglucanase.

Results

Pichia pastoris expressing endoglucanase, EG1, was grown in a 10 l fermenter yielding a high carboxymethyl cellulase (CMCase) activity of 340 U mg^?1. EG1-mediated modification of kraft pulp resulted in a paper sheet with the tensile index and burst index increased by 10 and 6.5 %, respectively. The kink index (indicating abrupt bends in fibres) of the enzyme-treated group decreased sharply by 45 % after the first recycling, compared with a reduction of only 1 % in the control group. Furthermore, EG1 treatment decreased the growth of crystallinity from 73.5 to 73.2 % and crystal size from 7.45 to 7.21 nm, which alleviated paper aging.

Conclusion

Endoglucanase EG1 modifies the interfacial properties of fibers, which affects fibre morphology during the recycling process and improves the technical properties of the resulting pulp and paper.

     

Identification and characterization of a novel bifunctional Δ<Superscript>12</Superscript>/Δ<Superscript>15</Superscript>-fatty acid desaturase gene from <Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis>

Objectives

To elucidate the biosynthesis pathway of linoleic acid and α-linolenic acid in Rhodosporidium kratochvilovae YM25235 and investigate the correlation of polyunsaturated fatty acids with its cold adaptation.

Results

A 1341 bp cDNA sequence, designated as RKD12, putatively encoding a Δ¹²-desaturase was isolated from YM25235. Sequence analysis indicated that this sequence comprised a complete ORF encoding 446 amino acids of 50.6 kDa. The encoded amino acid sequence shared higher similarity to known fungal Δ¹²-desaturases that are characteristic of three conserved histidine-rich motifs. RKD12 was further transformed into Saccharomyces cerevisiae INVScl for functional characterization. Fatty acid analysis showed the yeast transformants accumulated two new fatty acids: linoleic acid and α-linolenic acid. Furthermore, mRNA expression level of RKD12 and the content of linoleic acid and α-linolenic acid were increased significantly with the culture temperature downshift from 30 to 15 °C, which might be helpful for the cold adaptation of YM25235.

Conclusion

RKD12 is a novel bifunctional ?¹²/?¹⁵-desaturase gene, and the increased RKD12 mRNA expression level and PUFAs content at low temperature might be helpful for the cold adaptation of YM25235.

     

Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer

Objective

To investigate the effect of parthenolide on apoptosis and autophagy and to study the role of the PI3K/Akt signaling pathway in cervical cancer.

Results

Parthenolide inhibits HeLa cell viability in a dose dependent-manner and was confirmed by MTT assay. Parthenolide (6 µM) induces mitochondrial-mediated apoptosis and autophagy by activation of caspase-3, upregulation of Bax, Beclin-1, ATG5, ATG3 and down-regulation of Bcl-2 and mTOR. Parthenolide also inhibits PI3K and Akt expression through activation of PTEN expression. Moreover, parthenolide induces generation of reactive oxygen species that leads to the loss of mitochondrial membrane potential.

Conclusion

Parthenolide induces apoptosis and autophagy-mediated growth inhibition in HeLa cells by suppressing the PI3K/Akt signaling pathway and mitochondrial membrane depolarization and ROS generation. Parthenolide may be a potential therapeutic agent for the treatment of cervical cancer.

     

Molecular understandings on ‘the never thirsty’ and apomictic <Emphasis Type="Italic">Cenchrus</Emphasis> grass

The genus Cenchrus comprises around 25 species of ‘bristle clade’ grasses. Cenchrus ciliaris (buffel grass) is a hardy, perennial range grass that survives in poor sandy soils and limiting soil moisture conditions and, due to the very same reasons, this grass is one of the most prevalent fodder grasses of the arid and semi-arid regions. Most of the germplasms of Cenchrus produce seeds asexually through the process of apomeiosis. Therefore, the lack of sufficient sexual lines has hindered the crop improvement efforts in Cenchrus being confined to simple selection methods. Many attempts have been initiated in buffel grass to investigate the various molecular aspects such as genomic signatures of different species and genotypes, molecular basis of abiotic stress tolerance and reproductive performance. Even though it is an important fodder crop, molecular investigations in Cenchrus lack focus and the molecular information available on this grass is scanty. Cenchrus is a very good gene source for abiotic stress tolerance and apomixis studies. Biotechnological interventions in Cenchrus can help in crop improvement in Cenchrus as well as other crops through transgenic technology or marker assisted selection. To date no consolidated review on biotechnological interventions in Cenchrus grass has been published. Therefore we provide a thorough and in depth review on molecular research in Cenchrus focusing on molecular signatures of evolution, tolerance to abiotic stress and apomictic reproductive mechanism.