Fungal strain engineering from understanding towards applications

Abstract

Lignocellulosic biomass is an abundant and renewable resource, and has a promising potential as an alternative to fossil resources for industrial bioproduction of biofuels and value-added biochemicals. Filamentous fungi are the most important and efficient plant biomass degrading microorganisms and are widely used as cell factories in many industries. Fungal strain engineering has been applied for the development of more robust and versatile filamentous fungal cell factories, and considerable progress have been made in recent years, as described in Chapter 1. A thorough and comprehensive understanding of fungal physiological processes involved in plant biomass utilization is an essential prerequisite of rational and feasible strain engineering. Advances in omics technologies allow the important development of systems biology, and new genetic tools have been developed for improving the efficiency of genetic engineering of filamentous fungi, such as CRISPR/Cas9 technology. Chapter 2 showed that A. niger has the capacity to accumulate xylitol from lignocellulosic biomass and metabolic engineering is highly effective for the improvement of xylitol production in A. niger. This provides the industrial production of xylitol with an attractive alternative, as the direct use of lignocellulosic biomass by A. niger highly simplifies the xylitol bioproduction process. Besides, there are other crucial aspects in the process of xylitol production, which could be alternative targets for strain engineering, including the release of pentoses from lignocellulosic biomass and the transport of pentoses and polyols. The subsequent study showed that the manipulation of the xylanolytic transcriptional activator XlnR also effectively increased xylitol production from lignocellulosic biomass in A. niger. The transport of D-xylose was also considered to further stimulate xylitol production in Chapter 3. In addition to three characterized D-xylose transporters (XltA, XltB and XltC), a fourth D-xylose transporter (XltD) was identified in A. niger. XltD has similar efficiency as XltA, while XltB may be not a major D-xylose transporter under the tested conditions. The results also showed the existence of more D-xylose transporters in A. niger. Unfortunately, the modification of one D-xylose transporter in A. niger alone did not affect xylitol production, showing the complexity and redundancy of sugar transport system similar to sugar metabolism in A. niger. In Chapter 4, an L-arabitol transporter, LatA, was identified with high specificity for L-arabitol in A. niger and its homologs are widely present in Ascomycete fungi. Moreover, the deletion of latA positively affected L-arabitol production from wheat bran and sugar beet pulp, suggesting that this gene could be a target for the improvement of microbial cell factories. In Chapter 5, the interaction between three transcription factors GalX, GalR and AraR in D-galactose and L-arabinose catabolism was investigated in A. nidulans, revealing the involvement of all these regulators in D-galactose catabolism and the compensation phenomenon between different regulators. To summarize, the results of this thesis described different aspects of the physiology of Aspergillus species from metabolic and regulatory networks to sugar transport systems, which improve the understanding of these model fungi and facilitate the biotechnological applications of fungal cell factories for the production of valuable biochemicals.