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Kinetic transcriptome analysis reveals an essentially intact induction system in a cellulase hyper-producer Trichoderma reesei strain.

Poggi-Parodi D, Bidard F, Pirayre A, Portnoy T, Blugeon C, Seiboth B, Kubicek CP, Le Crom S, Margeot A - Biotechnol Biofuels (2014)

Bottom Line: Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated.The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei.Genes from two specific clusters might be potential targets for such genetic engineering.

View Article: PubMed Central - PubMed

Affiliation: IFP Energies nouvelles, 1-4 avenue de Bois-Préau, 92852 Rueil-Malmaison, France ; Sorbonne Universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), F-75005 Paris, France.

ABSTRACT

Background: The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour.

Results: We performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance.

Conclusions: This is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering.

No MeSH data available.


Related in: MedlinePlus

RUT C30 and NG 14 bioreactor culture profiles. Growth curves (dry biomass concentration g.L-1) and protein production levels (extracellular protein concentration g.L-1 (EC Prot. Conc.), as assayed by Bradford method (see Results), are displayed for NG 14 and RUT C30 T. reesei strains. Time 0 h marks the start of lactose feeding. Negative values represent the 24 h of batch culture and positive values the first 48 h of fed-batch culture with lactose. Each curve represents the average measure of two independent cultures; error bars show average standard deviation to give an estimate of replicates’ quality (excluding technical replicates). One NG 14 replicate is lacking after 24 h lactose induction, so dispersion data is not available. Bradford method allows accurate assessment of production start and comparison between strains but underestimates actual values and is therefore not appropriate for carbon balancing (see Results for detailed explanations).
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Fig1: RUT C30 and NG 14 bioreactor culture profiles. Growth curves (dry biomass concentration g.L-1) and protein production levels (extracellular protein concentration g.L-1 (EC Prot. Conc.), as assayed by Bradford method (see Results), are displayed for NG 14 and RUT C30 T. reesei strains. Time 0 h marks the start of lactose feeding. Negative values represent the 24 h of batch culture and positive values the first 48 h of fed-batch culture with lactose. Each curve represents the average measure of two independent cultures; error bars show average standard deviation to give an estimate of replicates’ quality (excluding technical replicates). One NG 14 replicate is lacking after 24 h lactose induction, so dispersion data is not available. Bradford method allows accurate assessment of production start and comparison between strains but underestimates actual values and is therefore not appropriate for carbon balancing (see Results for detailed explanations).

Mentions: The cultivation data for each fermentation are presented in (Additional file 1: Figure S1). Carbon source concentrations and biomass and protein production, focussed on the first 48 h after fed-batch are presented in Figure 1, with merged cultivation duplicates. CO2 production was monitored to achieve carbon balances, which reached more than 0.90 gCproduced/gCconsumed (data not shown). We chose to determine protein concentration using the Bradford method [31] to avoid background protein with peptides contained in the corn steep in the culture medium when protein concentration is low. However, for cellulases, this method is known to underestimate actual protein concentrations 3.5 to 5-fold [32] compared to the Lowry method [33], which reflects actual production. This conversion factor was used for mass balancing and to compare specific productivities with previous work (Jourdier et al. [29,30]). In addition, Lowry protein measurements were made on the RUT C30 cultivation that lasted for 150 h to check the consistency of the calculation (Additional file 2: Figure S2).Figure 1


Kinetic transcriptome analysis reveals an essentially intact induction system in a cellulase hyper-producer Trichoderma reesei strain.

Poggi-Parodi D, Bidard F, Pirayre A, Portnoy T, Blugeon C, Seiboth B, Kubicek CP, Le Crom S, Margeot A - Biotechnol Biofuels (2014)

RUT C30 and NG 14 bioreactor culture profiles. Growth curves (dry biomass concentration g.L-1) and protein production levels (extracellular protein concentration g.L-1 (EC Prot. Conc.), as assayed by Bradford method (see Results), are displayed for NG 14 and RUT C30 T. reesei strains. Time 0 h marks the start of lactose feeding. Negative values represent the 24 h of batch culture and positive values the first 48 h of fed-batch culture with lactose. Each curve represents the average measure of two independent cultures; error bars show average standard deviation to give an estimate of replicates’ quality (excluding technical replicates). One NG 14 replicate is lacking after 24 h lactose induction, so dispersion data is not available. Bradford method allows accurate assessment of production start and comparison between strains but underestimates actual values and is therefore not appropriate for carbon balancing (see Results for detailed explanations).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279801&req=5

Fig1: RUT C30 and NG 14 bioreactor culture profiles. Growth curves (dry biomass concentration g.L-1) and protein production levels (extracellular protein concentration g.L-1 (EC Prot. Conc.), as assayed by Bradford method (see Results), are displayed for NG 14 and RUT C30 T. reesei strains. Time 0 h marks the start of lactose feeding. Negative values represent the 24 h of batch culture and positive values the first 48 h of fed-batch culture with lactose. Each curve represents the average measure of two independent cultures; error bars show average standard deviation to give an estimate of replicates’ quality (excluding technical replicates). One NG 14 replicate is lacking after 24 h lactose induction, so dispersion data is not available. Bradford method allows accurate assessment of production start and comparison between strains but underestimates actual values and is therefore not appropriate for carbon balancing (see Results for detailed explanations).
Mentions: The cultivation data for each fermentation are presented in (Additional file 1: Figure S1). Carbon source concentrations and biomass and protein production, focussed on the first 48 h after fed-batch are presented in Figure 1, with merged cultivation duplicates. CO2 production was monitored to achieve carbon balances, which reached more than 0.90 gCproduced/gCconsumed (data not shown). We chose to determine protein concentration using the Bradford method [31] to avoid background protein with peptides contained in the corn steep in the culture medium when protein concentration is low. However, for cellulases, this method is known to underestimate actual protein concentrations 3.5 to 5-fold [32] compared to the Lowry method [33], which reflects actual production. This conversion factor was used for mass balancing and to compare specific productivities with previous work (Jourdier et al. [29,30]). In addition, Lowry protein measurements were made on the RUT C30 cultivation that lasted for 150 h to check the consistency of the calculation (Additional file 2: Figure S2).Figure 1

Bottom Line: Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated.The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei.Genes from two specific clusters might be potential targets for such genetic engineering.

View Article: PubMed Central - PubMed

Affiliation: IFP Energies nouvelles, 1-4 avenue de Bois-Préau, 92852 Rueil-Malmaison, France ; Sorbonne Universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), F-75005 Paris, France.

ABSTRACT

Background: The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour.

Results: We performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance.

Conclusions: This is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering.

No MeSH data available.


Related in: MedlinePlus