Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

The Patent Landscape of Polyhydroxyalkanoates Production by Algae and Cyanobacteria.

BACKGROUND: As global awareness regarding climate change and environmental pollution outcomes arise, eco-friendly and negative emission technologies emerge. METHODS: In this scenario, polyhydroxyalkanoate (PHA)-accumulating microorganisms play an important role in the transition from the petrochemical-based non-biodegradable polymer to renewable, eco-friendly, and biocompatible materials. More specifically, CO2 can be converted to biopolymers through photosynthesis by cyanobacteria and algae, posing as a promising technology for renewable material, CO2, and petroleum-dependence mitigations. However, although many microorganisms can accumulate PHA intracellularly, limitations persist, such as the elevated cost and limited market availability. RESULTS: Herein is presented a patent-based mapping on technological trends of PHAs production, including its production by microalgae and cyanobacteria using the Questel Orbit Intelligence software (version 1.9.8) in complement with the Espacenet Patent Search database. CONCLUSION: The inquiry on PHAs retrieved 34,243 patents filed since 1912, whereas 156 are related to their specific production by photosynthetic microorganisms, evidencing a prospective market for intellectual property.

Xylo-Oligosaccharide Utilization by Engineered Saccharomyces cerevisiae to Produce Ethanol.

The engineering of xylo-oligosaccharide-consuming Saccharomyces cerevisiae strains is a promising approach for more effective utilization of lignocellulosic biomass and the development of economic industrial fermentation processes. Extending the sugar consumption range without catabolite repression by including the metabolism of oligomers instead of only monomers would significantly improve second-generation ethanol production This review focuses on different aspects of the action mechanisms of xylan-degrading enzymes from bacteria and fungi, and their insertion in S. cerevisiae strains to obtain microbial cell factories able of consume these complex sugars and convert them to ethanol. Emphasis is given to different strategies for ethanol production from both extracellular and intracellular xylo-oligosaccharide utilization by S. cerevisiae strains. The suitability of S. cerevisiae for ethanol production combined with its genetic tractability indicates that it can play an important role in xylan bioconversion through the heterologous expression of xylanases from other microorganisms.

Evaluation of Product Distribution in Chemostat and Batch Fermentation in Lactic Acid-Producing Komagataella phaffii Strains Utilizing Glycerol as Substrate.

Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast Komagataella phaffii was genetically modified for the production of lactate from glycerol. For this, the bovine L-lactate dehydrogenase- (LDH)-encoding gene was inserted and the gene encoding the pyruvate decarboxylase (PDC) was disrupted, resulting in the GLp strain. This showed a yield of 67% L-lactic acid and 20% arabitol as a by-product in batches with oxygen limitation. Following up on these results, the present work endeavored to perform a detailed study of the metabolism of this yeast, as well as perturbing arabitol synthesis in an attempt to increase lactic acid titers. The GLp strain was cultivated in a glycerol-limited chemostat at different dilution rates, confirming that the production of both lactic acid and arabitol is dependent on the specific growth rate (and consequently on the concentration of the limiting carbon source) as well as on the oxygen level. Moreover, disruption of the gene encoding arabitol dehydrogenase (ArDH) was carried out, resulting in an increase of 20% in lactic acid and a 50% reduction in arabitol. This study clarifies the underlying metabolic reasons for arabitol formation in K. phaffii and points to ways for improving production of lactic acid using K. phaffii as a biocatalyst.

Differential effects of major inhibitory compounds from sugarcane-based lignocellulosic hydrolysates on the physiology of yeast strains and lactic acid bacteria.

OBJECTIVES: Major lignocellulosic inhibitory compounds found in sugarcane-based industrial hydrolysate samples were tested in laboratory and industrial yeast strains, as well as in lactic acid bacteria, in order to verify their effects on important physiological parameters. RESULTS: Saccharomyces cereviaise SA-1, an industrial strain, stood out as compared to the remaining strains for virtually all inhibitors investigated. This strain presented the highest growth rate and the lowest lag-phase in the presence of acetic acid, levulinic acid, p-coumaric acid, ferulic acid, and HMF, when compared to the other strains. In sugarcane-based hydrolysate fermentations, both SA-1 and CEN.PK113-7D presented similar fermentation performances. Industrial isolates of contaminating lactic acid bacteria were evaluated in the presence of an inhibitory cocktail, containing a mixture of 76.6 mM acetic acid, 1.3 mM HMF, 7.1 mM furfural, and 1.9 mM p-coumaric acid. Whilst all yeast strains were unable to grow under such conditions, bacteria had an average inhibition of roughly 50% on their growth rates. CONCLUSIONS: Overall, industrial strain SA-1 might be a promising microbial chassis for second generation ethanol production and for future metabolic and evolutionary engineering strategies, and for strain robustness understanding.