Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass

Lignocellulosic biomass is the most abundant renewable source of energy that has been widely explored as second-generation biofuel feedstock. Despite more than four decades of research, the process of ethanol production from lignocellulosic (LC) biomass remains economically unfeasible. This is due to the high cost of enzymes, end-product inhibition of enzymes, and the need for cost-intensive inputs associated with a separate hydrolysis and fermentation (SHF) process. Thermotolerant yeast strains that can undergo fermentation at temperatures above 40°C are suitable alternatives for developing the simultaneous saccharification and fermentation (SSF) process to overcome the limitations of SHF. This review describes the various approaches to screen and develop thermotolerant yeasts via genetic and metabolic engineering. The advantages and limitations of SSF at high temperatures are also discussed. A critical insight into the effect of high temperatures on yeast morphology and physiology is also included. This can improve our understanding of the development of thermotolerant yeast amenable to the SSF process to make LC ethanol production commercially viable.

Genome Shuffling of Mangrove Endophytic Aspergillus luchuensis MERV10 for Improving the Cholesterol-Lowering Agent Lovastatin under Solid State Fermentation

In the screening of marine mangrove derived fungi for lovastatin productivity, endophytic Aspergillus luchuensis MERV10 exhibited the highest lovastatin productivity (9.5 mg/gds) in solid state fermentation (SSF) using rice bran. Aspergillus luchuensis MERV10 was used as the parental strain in which to induce genetic variabilities after application of different mixtures as well as doses of mutagens followed by three successive rounds of genome shuffling. Four potent mutants, UN6, UN28, NE11, and NE23, with lovastatin productivity equal to 2.0-, 2.11-, 1.95-, and 2.11-fold higher than the parental strain, respectively, were applied for three rounds of genome shuffling as the initial mutants. Four hereditarily stable recombinants (F3/3, F3/7, F3/9, and F3/13) were obtained with lovastatin productivity equal to 50.8, 57.0, 49.7, and 51.0 mg/gds, respectively. Recombinant strain F3/7 yielded 57.0 mg/gds of lovastatin, which is 6-fold and 2.85-fold higher, respectively, than the initial parental strain and the highest mutants UN28 and NE23. It was therefore selected for the optimization of lovastatin production through improvement of SSF parameters. Lovastatin productivity was increased 32-fold through strain improvement methods, including mutations and three successive rounds of genome shuffling followed by optimizing SSF factors.

Breeding Actinobacillus succinogenes with Acid-tolerance by Genome Shuffling / 微生物学通报

A strain Actinobacillus succinogenes CGMCC 1593 was selected as the parent strain.After UV-EMS and UV-DES treatments respectively,seven mutated strains with subtle improvements in acid tol-erance were obtained,and were subjected for recursive protoplast fusion.Through three rounds of genome shuffling,four shuffled strains with both higher yield and acid tolerance were obtained.The shuffled strain namely F3-21 could even survive at pH 5.2.The comparison of the shuffled strains and the parent strain for succinic acid production was also studied here.After 48 h of shake-flask fermentation,the succinic acid concentration of F3-21 was 48% higher than that of the parent strain.When F3-21 was carried out in a 5 liter stirred bioreactor with pH controlled 5.6~6.0,the accumulation of succinic acid in 48 h fermentation attained 38.1 g/L,which was increased by 45% compared with that of the parent strain(26.2 g/L).While pH was controlled at 6.5~7.0,the production of succinic acid in 32 h fermentation attained 40.7 g/L.When F3-21 was carried out in fed-batch fermentation,succinic acid concentration of 67.4 g/L was reached in 72 h fer-mentation.These results indicated that the genome shuffling could improve the acid tolerance and the suc-cinic acid production of A.succinogenes CGMCC 1593.

Whole Genome Shuffling to Enhance Activity of Fibrinolytic Enzyme-producing Strains / 中国生物工程杂志

DC-12 is a Bacillus strain with strong fibrinolytic activity was screened from Douchi.A novel breeding technology——whole genome shuffling was applied to enhance the production of fibrinolytic enzyme.First of all,a candidate mutant library was constructed by treating DC-12 with ultraviolet rays andhNO2,respectively.Based on studying the optimum conditions for protoplast preparation and regeneration of DC-12,multi-parental whole genome shuffling with 4 strains was conceded by protoplasts electrical fusion,then combines with the screening method of double-inactive protoplast,two strains withhigher yield of fibrinolytic enzyme were obtained effectively,which can descend stably after five generations,and their enzyme production reached 2710 IU/ml,which was increased by 4~5 fold compared with the mutants,respectively.

Genome Shuffling and Its Prospect for Strain Improvement in Ethanol Production from Lignocellulosic Hydrolysates / 微生物学通报

Commercial production of bioethanol from lignocellulosic hydrolysates requires efficient fermenting strains. The abilities of the strain to converting all types of sugars in the hydrolysate to ethanol in high yield and to effectively tolerating/metabolizing inhibitors are necessary. Genome shuffling is a novel method for breeding, and it has been applied in pharmaceutical and food industry. This review summarized the technique of genome shuffling including principle, process, applications and its prospect for strains improvement in ethanol production from lignocellulosic hydrolysates.

Study of Breeding Saccharomyces cerevisiae with Improved Temperature and Ethanol Tolerance by Genome Shuffling / 微生物学通报

By UV induced mutagenesis of protoplasts of Saccharomyces cerevisiae strain f4, f5 and f6, and screening on plates containing different concertration of ethanol at different temperature, we obtained improved strains, such as f4.2, f5.1, f6.2, f4.5. By using a DES to deal with all the improved strains, We obtained two mutants, f5.1.1 and f4.2.1,which have improved ethanol torlerance. We made use of genome shuffling to generate improved strains in this work. We shuffled twice these improved strains by protoplast fusion and finally obtained strains with higher temperature and ethanol tolerance. we also identified shuffled strains that produced more ethanol by shake-flask experments. At the 35℃, the ethanol yield of R24 strain got to 12.93% (W/V), and were almost 5% higher than that of stain f4.