Screening Of Ethanol-Producing Strains With Pentose And Genomic Study Of Their Fermentation Mechanism

Nowadays,the energy crisis has been a common problem faced by human beings,fossil fuels are nearly depleting,and the bio-ethanol made from biomass,looms as one of the novel environmentally-friendly and renewable sources of energy,lignocellulose is becoming the most promising materials duo to its tremendous storage of the bio-energy.Apart from the energy from burning,the biomass energy research based on lignocellulose as raw materials mostly focuses on producing fuels by microbial fermentation,where the lignocellulose is hydrolyzed and the pentose in the end product was about 33%,of which 90%is xylose.However,pentose cannot be effectively used by most microbial strains.To fully utilize the sugar and improve the conversion rate of the biomass energy,proper microbial strains must be developed with great efforts.More than 100,000 species of microorganisms were known in nature,but only over 100 wild-type strains can directly use pentose.The screening and mechanism research of good strains lay the foundation for the engineering bacteria construction and the genetic improvement of the strains.In this paper,the growth characteristics of bacteria,yeasts and filamentous fungi were studied by bacteria screening,and low-yield strains were treated by UV and ⁶⁰Co-?ray radiation for induced mutation.Finally,two of differential strains from Fusarium oxysporum producing ethanol from pentose were selected,and then their transcriptomes were sequenced and analyzed by high-throughput sequencing techniques for differential strains at different fermentation stages.The mitochondrial genome of the high-ethanol yield strain mh2-2 was spliced and analyzed to obtain amounts of biological data,which provided experimental basis and data support for the subsequent efficient transformation work of microorganisms using lignocellulose to produce ethanol.The main research results are as follows:?1?18 strains in three types?four strains of yeast,one strain of bacteria,13 strains of filamentous fungi?were selected that were reported to be likely to produce ethanol with pentose and accharomyces cerevisia was chosen as control which effectively converts glucose into ethanol.All of the strains were from 111 strains of biomass saccharification/ethanol fermentation obtained by our laboratory for many years for pentose fermentation test.The results showed that CICC1960 Pichia stipitis and Enterobacter sp.showed a notable capability to produce ethanol in TTC screening.The results of pentose liquid fermentation test showed that the ethanol production of two strains was 4.088 g/L and 1.599/L respectively,in which the capability of Pichia stipitis was superior to other yeasts.In order to obtain the high-yield ethanol mutant to elevate the yield of ethanol,the yeast and Enterobacter sp.were induced by ⁶⁰Co-?ray radiation and UV for mutagenesis.The results showed that,⁶⁰Co-?ray radiation was more effective for Pichia stipitis with 15.6g/L in ethanol yield,about 4 times more than the ethanol produced by the original strain.The ethanol production did not witness remarkable changes in Enterobacter sp.after mutagenesis.In addition,among the filamentous fungi,the pentose liquid fermentation tests in 13 strains from different sources of Fusarium oxysporum showed that the ethanol production of Foc-mh2-2?M strain?was the highest,reaching 16.9 g/L,surpassing the high-yield mutant strain Pichia stipitis.In order to compare the transcriptomes of differential strains,Cs20?N strain?with an ethanol production of 2.8 g/L was selected as a relatively low ethanol yield strain,both M and N strains were Fusarium oxysporum,closely related in phylogeny,which provide ideal materials for subsequent comparative transcriptomics analysis.?2?Total RNAs were extracted from three phases,including the ethanol original phase?XM1 ethanol content?0.05 g/L?,accelerating phase?XM2?2g/L?and decline phase?XM3ethanol content reduction?0.1g/L?in M and N strains respectively as the samples to complete the transcriptome sequencing in Illumina Hiseq2500 sequencer,which generated over 4Gb data and the QC was more than 90%.Clean data were further filtered for the high quality data which were assembled by de novo with Trinity?version r20140717?,that is,the transcriptome data of six samples from each strain were assembled to obtain the transcript/gene sequence,with which the reads of each sample were aligned by BWA?version0.7.10?to the assembled transcript sequences;If the reads were uniquely aligned with its transcript sequence,the transcript would be expressed in this sample;the transcript/gene sequence in each sample was identified according to the alignment information.The spliced transcript/gene sequence was used to predict its protein sequence through the TransDecoder program in Trinity.Among the Trinity algorithm,homologous isoforms of many genes?including paralogues and variable spliced mRNA?were spliced,such as 19,070transcripts/genes encoding Cs20,in which 10,171 were unique transcripts/genes,of which3,752 unique transcripts/genes had at least 2 isoforms.Similarly,Foc-mh2-2 has 20,973transcripts/genes encoding proteins,in which 11,659 were unique transcripts/genes,of which 4,018 unique transcripts/genes had at least 2 isoforms.For the transcripts encoding the protein,the amino acid sequence was aligned in the EuKaryotic Orthologous Groups?KOG?database of NCBI.The threshold for BLASTP was 1e-5 during the analysis.?3?When the same strains were compared among different fermentation stages,it was found that the high-yield strain?M?and low-yield strain?N?shared a very significant differential gene cluster U,that is,the gene cluster for intracellular transport,secretion and vesicle transport.The results showed that the massive differential genes were annotated as ABC transporter in function,then we compared with those gene by Inparanoid Software Package between M and N,2 pairs of orthologous genes were found.These 2 pairs of genes were ABC transporters,and expressed in the decline phase in both of M and N strains.?4?Comparison of the expression of the genes in the metabolic pathways at different phases of M and N strains,respectively.The results showed that L-iditol dehydrogenase and xylulokinase were up-regulated in high-yield strain M and down-regulated in low-yield strain N,which played important roles in maintaining the metabolic flow towards ethanol production.?5?Comparison of The expression levels of differential strains in the same fermentation phase showed that among the three phases,M and N strains were significantly different in the expressed gene copies of the gene clusters for translation,ribosomal structure and biogenesis,and the gene clusters in replication,recombination and repair,among which the differential genes that only expressed in the high-yield strains rather than the low-yield strains were found.?6?Among the differential expression of M and N,many of the unique genes were annotated related to mitochondrion,during the biological process of pentose fermented to ethanol,the species requires plenty of energy that is mainly provided by mitochondrion.Obtained a complete mitogenome of Fusarium oxysporum mh2-2,with a size of 46 kb,which was different from sizes of any reported Fusarium mitogenomes,we have uploaded the sequence to NCBI GenBank accession number:MF155191.Comparative analysis of mh2-2 and other Fusarium mitogenomes we found that counts of hypothetical proteins and homing endonuclease genes?HEGs?do correlate with genomic sizes,and CRs and introns/IRs were the first and second factors contributing to size variation.Based on analysis of HEG clusters,orthologous groups,and their phylogenies,we hypothesized that the ancestor of Fusarium mitogenomes acquired these HEGs via horizontal transfer,and the mobility of introns/HEGs was independently transferred to several locations on ancestor mitogenomes,and then loss events happened for some genes during speciation and evolution,which resulted in size variation of mitogenomes.Although HEGs were defined as selfish genetic elements,we discovered company with physiological function,some of the HEGs were highly expressed suggesting mitochondrial genes play an role in gene function.Our results provided clues to further understanding of the functions and evolution of mitochondrial genes and genomes.