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Promoter Engineering Enables Overproduction of Foreign Proteins from a Single Copy Expression Cassette in Bacillus Subtilis

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Promoter Engineering Enables Overproduction of Foreign Proteins from a Single Copy Expression Cassette in Bacillus Subtilis Zhou et al. Microb Cell Fact (2019) 18:111 https://doi.org/10.1186/s12934-019-1159-0 Microbial Cell Factories RESEARCH Open Access Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis Chaoyang Zhou, Bin Ye, Shan Cheng, Leizhen Zhao, Yuanxin Liu, Jiandong Jiang and Xin Yan* Abstract Background: Bacillus subtilis is developed to be an attractive expression host to produce both secreted and cyto- plasmic proteins owing to its prominent biological characteristics. Chromosomal integration is a stable expression strategy while the expression level is not ideal compared with plasmid expression. Thus, to meet the requirement of protein overexpression, promoter, as one of the key elements, is important. It is necessary to obtain an ideal promoter for overproduction of foreign proteins from a single copy expression cassette. Results: The activity of promoter Pylb was further enhanced by optimizing the 35, 10 core region and upstream sequence (UP) by substituting both sequences with consensus sequences. The −fnal engineered− promoter exhibited almost 26-fold in β-galactosidase (BgaB) activity and 195-fold in super-folded green fuorescent protein (sfGFP) inten- sity than that of WT. The two proteins account for 43% and 30% of intracellular proteins, respectively. The promoter was eventually tested by successful extracellular overproduction of Methyl Parathion Hydrolase (MPH) and Chloro- thalonil hydrolytic dehalogenase (Chd) to a level of 0.3 g/L (144 U/mL) and 0.27 g/L (4.4 U/mL) on shake-fask culture condition. Conclusions: A strong promoter was engineered for efcient chromosomally integrated expression of heterologous proteins. Keywords: Bacillus subtilis, Promoter engineering, Chromosomal integration, Highly expression Background Plasmid-mediated recombinant production of pro- Bacillus subtilis, a species of Gram-positive aerobic soil teins in bacteria is unstable during the late stage of fer- bacteria, is an attractive industrial workhorse for pro- mentation [8]. Moreover, the safety concerns and legal duction of various enzymes and industrial recombinant requirements surrounding the use of antibiotic is another proteins due to its GRAS (generally recognized as safe) bottleneck in food industry. Chromosomal integration status, well-characterized protein secretion mechanisms ofers a more stable alternative to maintenance of for- and large-scale fermentation processes [1–5]. In addition, eign inserted expression cassette. However, the expres- the bacterium has no signifcant bias in codon usage and sion level may not meet the requirement when compared efcient genetic manipulation is available [6, 7]. Tus, with that of multi-copy plasmid expression. Protein pro- more attention has been paid to its expression systems duction was mainly determined by transcription, trans- for the purpose of the commercial application and basic lation and post-translation level. To realize efcient research. chromosomally integrated protein expression, promoter is of great importance in transcription level because it directly afects the efcient synthesis of fundamental *Correspondence: [email protected] Department of Microbiology, College of Life Sciences, Key Laboratory transcripts. Terefore, a powerful promoter is desirable for Microbiological Engineering of Agricultural, Environment of Ministry to drive gene overexpression. of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing 210095, Jiangsu, People’s Republic of China © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. Microb Cell Fact (2019) 18:111 Page 2 of 11 Nowadays, existing promoters can be generally divided promoters. A promoter with the consensus − 10 hex- into three major groups: constitutive promoters [9–13], amer (P10) was 0.5-fold stronger than Pylb while the BgaB inducible promoters [14–25] and stationary phase pro- expression level controlled by the P35 promoter resulted moters [26–28]. Recently, a stationary phase promoter in a sevenfold increase (Fig. 2b, c). When both changes Pylb [29] was identifed. Based on the Pylb promoter, in were combined into one single promoter (P3510), its this work, a new strong promoter was developed to real- activity was enhanced about ninefold (Fig. 2b, c and ize high level protein expression through single-copy Additional fle 1: Figures S3, S4). In addition to − 35 and expression cassette integration. − 10 regions, − 16 and − 22 region will also infuence promoter strength [30, 31]. Next, − 16 region was also Results changed into the corresponding consensus sequence Assessment of the WT promoter Pylb TRTG (where R stands for A or G) based on P3510, gen- To begin with, WT promoter was compared with com- erating P351016 (Fig. 2a). However, BgaB activity could monly used constitutive promoter P43 [9], inducible not be detected under the control of P351016 (Fig. 2b, promoter PxylA [14], and stationary phase promoter PsrfA c and Additional fle 1: Figures S3, S4). Te activity of [27]. Te expression level of reporter protein BgaB was promoter with mutation in the − 22 region (Fig. 2a) used to refect the strength of the promoters. Promoter decreased slightly under the control of P351022 (Fig. 2b, Pylb was prominent for its strength and stationary phase c and Additional fle 1: Figures S3, S4). (Fig. 1b, c and Additional fle 1: Figures S1, S2). During the lag phase and the early exponential phase, there was Engineering the upstream sequence to improve little BgaB activity detected. Te reporter protein began transcription to emerge at the mid-exponential phase; the activity Since upstream elements could enhance transcription sharply increased to the peak value during the transition initiation in B. subtilis [31, 32], to further enhance the to stationary phase, and remained constant during the promoter P3510, putative UP elements (− 59 to − 38) of followed stationary phase (Fig. 1c). Tus, the WT Pylb is four rrn operons (rrnO, rrnJ, rrnD, rrnB) controlled by deserved to be further engineered for overexpression of tandem promoters (P1 and P2) [33] were introduced to proteins in B. subtilis. replace the native region (Fig. 3a). As shown in Fig. 3b, four UP elements showed a distinct activation of the Engineering the core regions to improve transcription transcription as compared to the native UP element. Te − 35 and − 10 regions were the most important engineered BP3510 and JP3510 was approximately one sequences in promoter strength and the regions were fold stronger than that of the P3510 promoter, demon- widely engineered to enhance promoter transcrip- strating the strong stimulation of the promoter activity. tion [30]. Tus, the − 10 and − 35 regions of Pylb were When the UP element of rrnB was engineered to a con- changed into the corresponding consensus sequence sep- sensus sequence [34], − 59 nnAAA(A/T)(A/T)T(A/T) arately or in combination (Fig. 2a). Te reporter protein TTTTnnAAAAnnn − 38, the new UP element was BgaB was used to assess the strength of the engineered mutated to TTA AAA ATT TTT TTT AAA AAAA (Fig. 3a). ab c β-galactosidase activity lacA' Erm ori BGB pY P 8272 bp ylb bla bgaB lacA' P43 PxylA PsrfA Pylb Fig. 1 a Construction of the integrative plasmid pYBGB. b Comparison of the maximum yield of BgaB under the control of diferent promoters. All cultures were grown in triplicate, and each experiment was performed at least twice. Error bars indicate standard deviations. c The expression level and pattern of BgaB measured in strain WBBgaB. During 24 h of cultivation, cells were sampled periodically and analyzed by examining the biomass and BgaB activity Zhou et al. Microb Cell Fact (2019) 18:111 Page 3 of 11 Te mutant promoter NBP3510 showed superior activ- a nucleotide sequence of the core region ity to all of the other promoters and the BgaB expression -35 -22 -16 -10 was twofold higher than that of the P3510 (Fig. 3b, c and WT .....TTGGAT........AAT.....CGTTTACAAT..... Additional fle 1: Figures S5, S6) and enhanced by 26-fold compared with that of the WT promoter. In addition, the P35 .....TTGACA........AAT.....CGTTTACAAT..... native UP region of P3510 was also changed to a consen- sus sequence (Fig. 3a) and the resulting promoter NP3510 P10 .....TTGGAT........AAT.....CGTTTATAAT..... was enhanced up to 1.7-fold (Fig. 3b, c and Additional fle 1: Figures S5, S6). In summary, the strongest pro- P3510 .....TTGACA........AAT.....CGTTTATAAT..... moter NBP3510 allowed intracellular accumulation up to P351022 .....TTGACA........GGG.....CGTTTATAAT..... about 43% of the total cellular protein. In addition, pro- moter engineering had no signifcant efect on bacterial P351016 .....TTGACA........AAT.....ATGTTATAAT..... growth (Additional fle 1: Figures S14, S15 and Table S2). Tus, the increased production was mainly due to the b improved transcription. Western blot results showed that BgaB was expressed (Additional fle 1: Figures S11 and S13). Furthermore, qRT-PCR was used to verify the tran- scription level of NBP3510. RNA was extracted after 4, 8, 12, 16 h. Te highest transcription level of NBP3510 was 340-fold stronger than promoter Pylb (Fig. 3d). Although the strength of NBP3510 was dramatically enhanced at both log phage and stationary phase after engineering, it still exhibits the property of “station- ary phase” (Fig. 1c and Additional fle 1: Figures S5, S6). Since target gene was also transcribed in the early stage of cell growth under the control of stationary-phase pro- moter. Tus, the kinetics of BgaB production were not ft very well with Luedeking and Piret [35] (Additional fle 1: Figure S17 and Table S3).
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