Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose.

All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growth-based screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.

Novel strategies to improve co-fermentation of pentoses with D-glucose by recombinant yeast strains in lignocellulosic hydrolysates.

Economically feasible production of second-generation biofuels requires efficient co-fermentation of pentose and hexose sugars in lignocellulosic hydrolysates under very harsh conditions. Baker's yeast is an excellent, traditionally used ethanol producer but is naturally not able to utilize pentoses. This is due to the lack of pentose-specific transporter proteins and enzymatic reactions. Thus, natural yeast strains must be modified by genetic engineering. Although the construction of various recombinant yeast strains able to ferment pentose sugars has been described during the last two decades, their rates of pentose utilization is still significantly lower than D-glucose fermentation. Moreover, pentoses are only fermented after D-glucose is exhausted, resulting in an uneconomical increase in the fermentation time. In this addendum, we discuss novel approaches to improve utilization of pentoses by development of specific transporters and substrate channeling in enzyme cascades.

Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels.

Bioalcohols produced by microorganisms from renewable materials are promising substitutes for traditional fuels derived from fossil sources. For several years already ethanol is produced in large amounts from feedstocks such as cereals or sugar cane and used as a blend for gasoline or even as a pure biofuel. However, alcohols with longer carbon chains like butanol have even more suitable properties and would better fit with the current fuel distribution infrastructure. Moreover, ethical concerns contradict the use of food and feed products as a biofuel source. Lignocellulosic biomass, especially when considered as a waste material offers an attractive alternative. However, the recalcitrance of these materials and the inability of microorganisms to efficiently ferment lignocellulosic hydrolysates still prevent the production of bioalcohols from these plentiful sources. Obviously, no known organism exist which combines all the properties necessary to be a sustainable bioalcohol producer. Therefore, breeding technologies, genetic engineering and the search for undiscovered species are promising means to provide a microorganism exhibiting high alcohol productivities and yields, converting all lignocellulosic sugars or are even able to use carbon dioxide or monoxide, and thereby being highly resistant to inhibitors and fermentation products, and easy to cultivate in huge bioreactors. In this review, we compare the properties of various microorganisms, bacteria and yeasts, as well as current research efforts to develop a reliable lignocellulosic bioalcohol producing organism.