Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01

Abstract

Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA.

Figure 1

Recombinant plasmids for promoter characterization with gfp as a reporter gene. (a) Recombinant plasmid with a lac promoter as a control. (b) Recombinant plasmids for characterizing the strengths of 10 selected endogenous promoters.

Figure 2

Characterization of the chosen promoters and lac promoter via qPCR analysis. Transcription of gfp gene under different promoters in P. mendocina NKU was quantified at different growth phases. 16S rDNA gene was used as internal reference. The relative transcription value of gfp gene under lac promoter was set as 1. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between lac promoter and chosen promoters. * and ** indicate P < 0.05 and P < 0.01, respectively.

Figure 3

Characterization of the chosen promoters and lac promoter via GFP fluorescence intensity measurements. Expression of gfp gene under different promoters in P. mendocina NKU was quantified at different growth phases. The background expression was subtracted, and the relative fluorescence intensity was calculated by normalization against per OD₆₀₀ of whole cells. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between lac promoter and chosen promoters. * and ** indicate P < 0.05 and P < 0.01, respectively.

Figure 4

Characterization of the chosen promoters and lac promoter via confocal microscope. (A) Green fluorescence within the cell. (B) Outline of cell membrane by stain with FM4-64/L. (C) A and B merged together. All the images were taken at the same exposure condition.

Figure 5

The construction schematic diagram for inserting the promoters into upstream of phaC1 gene and for knockout of phaZ in the genome of P. mendocina NKU.

Figure 6

qPCR analysis and PHA fermentation results for the strains NKU-4C1, NKU-6C1, NKU-16C1 and NKU. Transcriptional levels of phaC1 (a), phaC2 (b) and phaZ (c) for the different strains. (d) Cell dry weight (CDW) and PHA production for the strains. Samples for qPCR were taken at 36 h of PHA fermentation. The transcriptional level for strain NKU was set as 1. wt% was defined as the ratio of PHA to CDW. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between NKU and the mutants. * and ** indicate P < 0.05 and P < 0.01, respectively.

Figure 7

qPCR analysis and PHA fermentation results for the strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1, NKU-∆phaZ-16C1 and NKU-∆phaZ. Transcriptional levels of phaC1 (a), phaC2 (b) and phaZ (c) for the different strains. (d) CDW and PHA production for the strains. Samples for qPCR were taken at 36 h of PHA fermentation. The transcriptional level for strain NKU-∆phaZ was set as 1. Data represent the mean values ± standard deviations of triplicate measurements from three independent experiments. A Student’s t-test was performed between NKU-∆phaZ and other mutants. * and ** indicate P < 0.05 and P < 0.01, respectively.