Screening of a genome-reduced Corynebacterium glutamicum strain library for improved heterologous cutinase secretion

Abstract

The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome-reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High-throughput phenotyping using robotics-integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass-specific cutinase yield for strain GRS41_51_NprE was increased by ~ 200%, although the growth rate was reduced by ~ 60%. Importantly, the causative deletions of genomic clusters cg2801-cg2828 and rrnC-cg3298 could not have been inferred a priori. Strikingly, bioreactor fed-batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a 'high-performance' strain from batch screening into a 'low-performance' strain in fed-batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C. glutamicum to both genomic deletions and different bioprocess conditions.

Fig. 1

Observed growth rates µ and biomass‐specific cutinase yields Y _P/X from C. glutamicum GRS library screening, normalized with respect to phenotype of the respective reference strain. (A) Relative GRS‐specific growth rates µ ^GRS/µ ^WT. (B) Relative GRS‐specific cutinase yields Y _P/X ^GRS/Y _P/X ^WT. Each bar represents one GRS, and the Sec SP used is indicated by different colours. Error bars indicate standard deviation from growth experiments conducted in eleven to 46 replicates, as indicated in Table S1. GRSs were cultivated aerobically with 20 g l⁻¹ d‐glucose in CGXII‐defined mineral medium, using an LHS with integrated MBR system. Cultivations took place in 48‐well FlowerPlates (800 µl culture volume, 30 °C, 1300 rpm). Cultivations were automatically harvested shortly after transition from exponential to stationary phase. GRS, genome‐reduced strain. SP, signal peptide. LHS, liquid handling system. MBR, microbioreactor.

Fig. 2

Correlation of biomass‐specific cutinase yields Y _P/X with growth rates µ for the cutinase secretion GRS library. The overall best performing strain GRS41_51_NprE is indicated, as well as its counterpart using the AmyE SP for cutinase secretion. For comparison, the corresponding reference strains WT_NprE and WT_AmyE are also indicated. Each data point represents one GRS, and the Sec SP used is indicated by different colours. Error bars indicate standard deviation from growth experiments conducted in eleven to 46 cultivation replicates, as indicated in Table S1. GRS, genome‐reduced strain. SP, signal peptide.

Fig. 3

Impact of single gene cluster deletions (upper row) and combinations thereof (lower row) on growth and cutinase secretion phenotypes for resulting GRSs. Subscripts of bar plots indicate relevant single gene cluster deletion of strains GRS21_NprE, GRS41_NprE and GRS51_NprE (upper row, from left to right), as well as combinatorial deletion of gene clusters for strains GRS21_41_NprE and GRS41_51_NprE (lower row, left and right respectively). For strain GRS41_51_NprE, the specific combination of deleting clusters cg2801‐cg2828 and rrnC‐cg3298 led to the strongly increased biomass‐specific cutinase yield. This specific combination was not obvious from the other data obtained. Strains are based on CR099; for details, see Table 1. Bar plots show growth rate µ (light grey) and biomass‐specific cutinase yield Y _P/X (dark grey); for corresponding values, see Table S1. GRS, genome‐reduced strain.

Fig. 4

Effect of bioprocess conditions on substrate‐specific cutinase yield Y _P/S and biomass‐specific cutinase yield Y _P/X for strain GRS41_51_NprE and reference strain WT_NprE. Arrows indicate relative changes in parameter values from strain WT_NprE (light grey bars) to GRS41_51_NprE (dark grey bars). Data for fed‐batch cultivations with WT_NprE are taken from the literature (Hemmerich et al., 2019a). Bioreactor fed‐batch cultivations were conducted using a glucose feeding function calculated with feeding profile parameter µ _Set = 0.15 h⁻¹. IPTG to induce recombinant gene expression was added to a final concentration of 50 µM at the beginning of the fed‐batch phase. MBR, microbioreactor. STR, stirred tank reactor.