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.

Deletion of genomic islands in the Pseudomonas putida KT2440 genome can create an optimal chassis for synthetic biology applications

“…Transcriptome analysis was carried out by using a standard pipeline. The detailed analysis process referred to a previous literature [35].…”

“…After the fermentation was completed, the bacterial cells were collected by centrifugation at 13,000g and 4 °C for 20 min, lyophilized and weighed. PHA extraction and purification were performed according to the previous methods [35].…”

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production

“…Promoter engineering is considered as a promising approach for enhanced production of bacterial secondary metabolites [21,22]. FPKM (fragments per kilobase million) value is positively correlated with the transcriptional activity of a gene [40], which therefore can be regarded as an indicator for initial screening of promoters.…”

“…Currently, endogenous promoters are highlighted as promising candidates for improved production of bacterial secondary metabolites [21]. For example, 14 endogenous promoters identi ed from Streptomyces albus J1074 by RNA-seq and reporter assays were successfully used to activate a cryptic gene cluster in S. griseu [22].…”

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production

“…Currently, endogenous promoters are highlighted as promising candidates for improved production of bacterial secondary metabolites [ 21 ]. For example, 14 endogenous promoters identified from Streptomyces albus J1074 by RNA-seq and reporter assays were successfully used to activate a cryptic gene cluster in S. griseu [ 22 ].…”