Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host

Abstract

The impact of overproduction of a heterologous protein on the metabolic system of host was investigated. The protein expression profiles of IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

The Effect of Recombinant Protein Production in Lactococcus lactis Transcriptome and Proteome

“…The attempts to increase pDNA and recombinant protein production in L. lactis have a direct repercussion in its transcriptome, proteome and metabolome, when compared with a wild-type strain [29][30][31]. The findings from such studies are extremely useful for the effectiveness of synthetic biology methodologies, wherein a rational engineering approach of the L. lactis genome (gene knockout and/or overexpression) would increase/optimize the pDNA and recombinant protein production, in terms of both yield and quality [28].…”

“…It was previously described that the number of plasmid copies per cell (or plasmid copy number, PCN) could influence the expression level of some proteins [46], which in turn leads to alterations in the cell proteome profile. In a recent study (Figure 7, compiled data from [29]), the GFP gene under the control of the strong inducible promoter nisA was cloned into the low PCN pHR086 plasmid (6-9 copies per cell) and into the high PCN pJH24 plasmid (45-85 copies per cell), both derived from pIL252 with pAMβ1-based replicons [29]. As a result, the high PCN plasmid expressed 4-fold more GFP than its low PCN counterpart, making GFP the most abundant protein in the bacterial proteome [29].…”

“…In a recent study (Figure 7, compiled data from [29]), the GFP gene under the control of the strong inducible promoter nisA was cloned into the low PCN pHR086 plasmid (6-9 copies per cell) and into the high PCN pJH24 plasmid (45-85 copies per cell), both derived from pIL252 with pAMβ1-based replicons [29]. As a result, the high PCN plasmid expressed 4-fold more GFP than its low PCN counterpart, making GFP the most abundant protein in the bacterial proteome [29]. Several stress response and chaperone proteins (groES, groEL trxB1, grpE, nusG and dnaJ), as well as ATP synthase, were up-regulated in the high PCN strain to increase protein synthesis and stability, and to allow the cell to cope with the stress of having an increase in the plasmid maintenance costs, while the enzymes of the amino acid pathway (de novo synthesis and interconversion of amino acids) were downregulated [29].…”

“…As a result, the high PCN plasmid expressed 4-fold more GFP than its low PCN counterpart, making GFP the most abundant protein in the bacterial proteome [29]. Several stress response and chaperone proteins (groES, groEL trxB1, grpE, nusG and dnaJ), as well as ATP synthase, were up-regulated in the high PCN strain to increase protein synthesis and stability, and to allow the cell to cope with the stress of having an increase in the plasmid maintenance costs, while the enzymes of the amino acid pathway (de novo synthesis and interconversion of amino acids) were downregulated [29]. Several glycolytic enzymes and transcription/translation-related proteins were also highly expressed.…”

“…All these alterations were much more significant when using the high PCN plasmid compared with the low PCN counterpart. A major difference found in the high PCN strain was in the glucose metabolism, with some enzymes belonging to the sugar nucleotide metabolism being up-(pfl and adhE) or down-regulated (zwf and gnd-pentose phosphate pathway) [29]. If some pentose phosphate pathway enzymes were down-regulated, the carbon flow for purine and pyrimidine synthesis should have decreased.…”