Metabolic engineering of Escherichia coli for efficient

Vol:.(1234567890)

Systems Microbiology and Biomanufacturing (2021) 1:444–458https://doi.org/10.1007/s43393-021-00031-1

1 3

ORIGINAL ARTICLE

Metabolic engineering of Escherichia coli for efficient ectoine production

Shuyan Zhang1,2 · Yu Fang1,2 · Lifei Zhu1,2 · Hedan Li1,2 · Zhen Wang1,2 · Ying Li1,2 · Xiaoyuan Wang1,2,3

Received: 7 March 2021 / Revised: 10 April 2021 / Accepted: 15 April 2021 / Published online: 14 May 2021 © Jiangnan University 2021

AbstractEctoine is a high-value stabilizer and protective agent with various applications in enzyme industry, cosmetics, and biomedi-cine. In this study, rational engineering strategies have been implemented in Escherichia coli to efficiently produce ectoine. First, the synthetic pathway of ectoine was constructed in E. coli MG1655 by introducing an artificial thermal switch system harboring the ectABC cluster from Halomonas elongate, and the resulting strain produced 1.95 g/L ectoine. Second, crr encoding the glucose-specific enzyme II domain A of phosphotransferase system and iclR encoding the glyoxylate shunt transcriptional repressor were deleted in E. coli for enhancing the oxaloacetate supply, leading to the increasement of the ectoine titer to 9.09 g/L. Third, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was removed from the genome to weaken the competitive pathway; simultaneously, an endogenous feedback-resistant lysC was overexpressed to complement the enzymatic activity deficiency of the aspartate kinase, leading to 30.36% increase of ectoine titer. Next, the expression of phosphoenolpyruvate carboxylase was modulated with varying gradient strength promoters to accelerate the biosynthesis efficiency of ectoine. Finally, aspDH encoding aspartate dehydrogenase from Pseudomonas aeruginosa PAO1 was overexpressed to further improve the biosynthesis of ectoine. The final strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 produced 30.37 g/L ectoine after 36-h fed-batch fermentation with a yield of 0.132 g/g glucose and a productivity of 0.844 g/(L h).

Keywords Escherichia coli · Rational engineering · Ectoine production · Precursor accumulation · PaeAspDH · Temperature-controlled fermentation

Introduction

Ectoine, a heterocyclic amino acid, was first detected in the extremely halophilic phototrophic bacterium Ectothiorho-dospira halochloris [1]. It can mitigate noxious effects on nucleic acids, proteins and viable cells brought by dehydra-tion, radiation, freeze-thawing, high osmotic pressure, and

chemical reagent [2]. Due to its protective properties, which can be transferred to human skin, ectoine has been widely added in cosmetic products [3]. Recently ectoine has also attracted increasing interest because of its potential medici-nal value in human diseases, such as Alzheimer’s disease [4], lung inflammation [5], and colitis [6]. It is estimated that the global production level of ectoine is approximately 15,000 tons per annum, with a putative sales value of about 1000 US dollars/kg [7]. Therefore, ectoine, as a promising high-value chemical, has drawn considerable attention due to its multiple applications and large market demand.

The increasing commercial demand for ectoine has resulted in numerous efforts to improve its production. Chemical synthesis of ectoine can be achieved, but the pro-cess is complex and costly if high purity and stereoselectiv-ity is requested [8]. Under stress conditions, microorgan-isms such as Halomonas elongate [9] and Chromohalobacter salexigens [10] synthesize ectoine naturally inside the cells as a protectant. However, these bacteria require fermentation

Shuyan Zhang and Yu Fang contributed equally to this work.

* Xiaoyuan Wang [email protected]

1 State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China

2 Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

3 International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, China

http://crossmark.crossref.org/dialog/?doi=10.1007/s43393-021-00031-1&domain=pdf

445Systems Microbiology and Biomanufacturing (2021) 1:444–458

1 3

conditions that impose stress on the cells to trigger tran-scription of the genes relative to ectoine synthesis, when industrially producing ectoine. A discontinuous harvest-ing procedure called “bacterial milking” was established for large-scale production of ectoine using H. elongate [9], which involves salt shock, thus increasing reactor corrosion, reducing volumetric yield and complicating downstream processing [11]. Great efforts have been made to avoid the drawbacks of high salinity media. Several mutants of moder-ate halophiles H. elongate with ectoine hyper-synthesizing ability were constructed [12, 13]. In addition, Zhang et al. utilized an ectoine-excreting strain Halomonas salina DSM 5928 to produce the compatible solute at relatively low NaCl concentration [14].

After discovering the ectoine biosynthetic cluster, the ectoine biosynthetic pathway has been constructed in non-halophilic bacteria, such as Escherichia coli [11] and Corynebacterium glutamicum [15, 16]. Beginning with pre-cursor l-aspartate-β-semialdehyde, the ectoine biosynthesis pathway consists of three enzymatic steps as shown in Fig. 1.

First, l-aspartate-β-semialdehyde is conversed into l-2,4-di-aminobutyrate (DABA) by l-2,4-diaminobutyrate transami-nase encoded by ectB. Subsequently, DABA is acetylated to N-γ-acetyldiaminobutyric acid (NADA) by l-diaminobutyric acid acetyl transferase, encoded by ectA. Finally, ectoine syn-thase, encoded by ectC catalyzes the conversion of NADA into ectoine [8]. The expression ratio among the three key enzymes was optimized through transcriptional balancing to increase ectoine synthesis capacity [17]. By the whole-cell biocatalysis, the recombinant E. coli in which the non-native ectoine synthesis pathway was constructed produced ectoine with high efficiency using aspartate and glycerol as the direct substrates [18]. Besides, the AraC-ect biosensor, engineered from AraC to recognize ectoine as its non-natural effector, was used in high throughput screening of ectoine overproducing strains [19]. Recently, E. coli W3110 was transformed to an ectoine-producing strain by system metabolic engineering, of which the titer and productivity of ectoine reached 25.1 g/L and 0.84 g/(L h), respectively [20]. By “stimulus response-based flux-tuning” method, Halomonas bluephagenesis has

Fig. 1 Metabolic engineering E. coli for ectoine production. Straight dash lines indicate the elimination of transcriptional repression. Curved dash lines suggest the blocking up of the reactions. Bold lines represent the enhanced enzymatic reactions. Genes encoding for the corresponding metabolic enzymes or transporters are shown in italic next to the reaction arrows. The genes with crosses beside them are deleted from the genome of E. coli. The genes in boxes are overex-pressed by high copy plasmids. crr encodes glucose-specific enzyme II domain A of the glucose-specific phosphotransferase system; iclR encodes the transcriptional repressor of aceB and aceA; aceB and

aceA encode malate synthase A and isocitrate lyase, respectively; ppc encodes phosphoenolpyruvate decarboxylase; aspC encodes endogenous aspartate aminotransferase; aspDH encodes L-aspartate dehydrogenase from P. aeruginosa PAO1; thrA and metL encode the bifunctional aspartokinase/homoserine-dehydrogenase; EclysC* and CglysC* are feedback-resistant genes encoding aspartokinase from E. coli and C. glutamicum, respectively; ectA encodes L-2,4-diam-inobutyrate acetyltransferase; ectB encodes L-2,4-diaminobutyrate transaminase; ectC encodes ectoine synthase

446 Systems Microbiology and Biomanufacturing (2021) 1:444–458

1 3

also been chromosomally engineered for enhanced production of ectoine from glucose [21].

It is either by galactose permease or glucose-specific phos-photransferase system (PTS) that glucose can be transported into the E. coli cell. PEP is needed for the transport of glucose by PTS [22]. It is reported that the deletion of the gene crr, which encodes the glucose-specific enzyme II domain A of PTS, could increase the accumulation of PEP, resulting in the enhancement of l-threonine accumulation [23]. When iclR, a glyoxylate shunt transcriptional repressor gene, is knocked out, the glyoxylate bypass can be enhanced, accumulating oxaloacetate precursor to produce l-threonine [24], ectoine [20] and 3-aminopropionic acid [25]. The gene thrA encodes the bifunctional aspartokinase/homoserine dehydrogenase, of which the deletion can lead to the increase of ectoine produc-tion. The enzymatic activity deficiency of the aspartate kinase caused by thrA deletion can be compensated by overexpression of feedback-resistant lysC, which also encodes aspartokinase [20]. It is well documented that phosphoenolpyruvate carboxy-lase encoded by the gene ppc is the key enzyme catalyzing the biosynthesis of oxaloacetate [26], and the expression of ppc has an optimal level for l-threonine production [27]. As previ-ously reported, aspartate dehydrogenase encoded by aspDH from Pseudomonas aeruginosa PAO1 can aminate oxaloac-etate directly to form aspartate [28]. This enzyme has been heterologously introduced to E. coli to enhance the accumula-tion of aspartate and promote the production of β-alanine [26].

In this study, a 100% genetically defined strain capable of synthesizing ectoine efficiently was constructed starting from a model strain E. coli MG1655 by rational metabolic engineering strategies, and fermentation production was designed to efficiently produce ectoine by temperature-regulated promoters without adding any inducer. First, the ectABC cluster from H. elongata ligated in a thermal switch system was introduced into MG1655, and the resultant strain was capable to produce ectoine. Inspired by several meta-bolic engineering strategies for l-aspartate family amino acids and derivatives [29, 30], the metabolic pathway of the engineered strain was rationally refitted to improve its capability of producing ectoine by deleting crr, iclR, and thrA, overexpressing endogenous lysC and ppc as well as exogenous aspDH from P. aeruginosa PAO1. The final constructed strain, named as MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3, synthesized 30.37 g/L ectoine by fed-batch fermentation.

Materials and methods

Bacterial strains and plasmids

All bacterial strains and plasmids used in this study are shown in Table 1. The model strain of E. coli, MG1655,

was utilized as the parent strain and genetically engineered to synthesize ectoine. E. coli JM109 was used for construct-ing recombinant plasmids. The plasmid pCas and pTargetF were applied for deletion of genes on the genome based on CRISPR-Cas9 system [31], and pFT28 vector was used to express the ectABC cluster and other key enzyme encoding genes.

Recombinant plasmid construction

All the primers used in this study are listed in the Table 2.The plasmid pTargetF-crr was derived from the original

pTargetF by inverse PCR, using the primers crr-sgRNA-F and crr-sgRNA-R. The PCR product was digested with DpnI to remove the template plasmid, and transformed into E. coli JM109 to form circular plasmid. Other plasmids pTargetF-iclR, pTargetF-thrA and pTargetF-aspC were constructed in the same way with different primers.

Plasmid pFT28 was reconstructed from pFT24 which was originally constructed by Fang et al. [32] by remov-ing tetR and the PLtetO1 promoter as well as replacing the p15A replicon with pMB1 replicon that confers a high copy number. The genetic manipulations described above were accomplished using ClonExpress II One Step Cloning Kit (Vazyme, Jiangsu, China).

After adjusting the codon usage to that preferred by E. coli, the codon-optimized 2433-bp ectABC gene cluster (GeneBank accession number: MW316739), according to the genome of H. elongate, was synthesized by TIANLIN Bio Inc (Wuxi, China). The synthesized gene was amplified with primers to which appended a high strength RBS and then inserted into SacI site of pFT28 located downstream of the pR-pL promoter by One Step Cloning Kit, constructing the plasmid pFT28-ectABC.

A 1028-bp DNA fragment was obtained using the primers lysC-cg2 (including a mutated base C932T) and lysC-cg1 (including a mutated base G1A and appended a PR pro-moter as well as a high strength RBS) from C. glutamicum ATCC 13032. The primers lysC-cg3 (including a mutated base C932T) and lysC-cg4 were used to amplify the other 395-bp DNA fragment. The complete 1404-bp fragment was amplified with lysC-cg1 and lysC-cg4 as the primers and the mixture of the two DNA fragments as the template by overlapping PCR. By One Step Cloning Kit, the 1404-bp fragment was ligated into KpnI site of pFT28-ectABC to construct pFT28-ectABC-CglysC*. With the same method, the plasmid pFT28-ectABC-EclysC* was constructed using lysCec1F, lysCec1R, lysCec2F and lysCec2R as primers and the genome of MG1655 as a template.

Based on the genome of P. aeruginosa PAO1, the prim-ers aspDH-F added the PR promoter and aspDH-R were designed and used to amplify the aspartate dehydrogenase encoding gene aspDH. The PCR product was purified and


1 3

Table 1 Bacterial strains and plasmids used in this study

Strain/plasmid Description Source

Strains JM109 Wild-type E. coli NEB MG1655 Wild-type E. coli CGSC MWZ001 MG1655Δcrr This study MWZ002 MG1655ΔcrrΔiclR This study MWZ003 MG1655ΔcrrΔiclRΔthrA This study MWZ004 MG1655ΔcrrΔiclRΔthrAΔaspC This study MG1655/pFT28-ectABC MG1655 harboring the plasmid pFT28-ectABC This study MWZ001/pFT28-ectABC MWZ001 harboring the plasmid pFT28-ectABC This study MWZ002/pFT28-ectABC MWZ002 harboring the plasmid pFT28-ectABC This study MWZ003/pFT28-ectABC MWZ003 harboring the plasmid pFT28-ectABC This study MWZ002/pFT28-ect-

ABC-EclysC*MWZ002 harboring the plasmid pFT28-ectABC-EclysC* This study

MWZ002/pFT28-ect-ABC-CglysC*

MWZ002 harboring the plasmid pFT28-ectABC-CglysC* This study

MWZ003/pFT28-ect-ABC-EclysC*

MWZ003 harboring the plasmid pFT28-ectABC-EclysC* This study

MWZ003/pFT28-ect-ABC-CglysC*

MWZ003 harboring the plasmid pFT28-ectABC-CglysC* This study

MWZ003/pFT28-ect-ABC-EclysC*-ppc1

MWZ003 harboring the plasmid pFT28-ectABC- EclysC*-ppc1 This study





MWZ003/pFT28-ect-ABC-EclysC*-aspDH

MWZ003 harboring the plasmid pFT28-ectABC- EclysC*-aspDH

This study

MWZ003/pFT28-ect-ABC-EclysC*-aspC

MWZ003 harboring the plasmid pFT28-ectABC- EclysC*-aspC This study

MWZ004/pFT28-ect-ABC-EclysC*-aspC

MWZ004 harboring the plasmid pFT28-ectABC- EclysC*-aspC This study

MWZ004/pFT28-ect-ABC-EclysC*-aspDH

MWZ004 harboring the plasmid pFT28-ectABC- EclysC*-aspDH

This study

Plasmids pCas repA101(Ts) kan Pcas-cas9 ParaB-Red lacIq Ptrc-sgRNA-pMB1 [31] pTargetF pMB1 aadA sgRNA, spectinomycin-resistant [31] pTargetF-crr pMB1 aadA sgRNA-crr, spectinomycin-resistant This study pTargetF-iclR pMB1 aadA sgRNA-iclR, spectinomycin-resistant This study pTargetF-thrA pMB1 aadA sgRNA-thrA, spectinomycin-resistant pMB1 aadA

sgRNA-aspC, spectinomycin-resistantThis study

pTargetF-aspC Triclosan-resistant, p15A ori, λcI (ts), PRL::tetR, MCS1, PLtetO1::MCS2

This study

pFT24 Derived from pFT24, removing tetR and PLtetO1 as well as replac-ing p15A with pMB1 replicon

[32]

pFT28 Derived from pFT28, inserting ectABC gene cluster This study pFT28-ectABC Derived from pFT28-ectABC, inserting feedback-insensitive lysC

(C1055T) from E. coli under the control of PR

This study

pFT28-ectABC-EclysC* Derived from pFT28-ectABC, inserting feedback-insensitive lysC (G1A, C932T) from C. glutamicum under the control of PR

This study

pFT28-ectABC-CglysC* Derived from pFT28-ectABC-EclysC*, inserting ppc under the control of the promoter PJ23100

This study

pFT28-ectABC-EclysC*-ppc1

Derived from pFT28-ectABC-EclysC*, inserting ppc under the control of the promoter PJ23107

This study


1 3

integrated into pFT28-ectABC-EclysC* digested with XhoI restriction enzyme, yielding pFT28-ectABC-EclysC*-aspDH. Similarly, the plasmid pFT28-ectABC-EclysC*-aspC was constructed by ligating the PCR product, which was amplified with the primers aspC-F added the PR pro-moter and aspC-R according to the genome of E. coli, into pFT28-ectABC-EclysC* digested with XhoI restriction enzyme.

With PJ23100-ppc-F, PJ23107-ppc-F, PJ23115-ppc-F and ppc-R as primers and MG1655 genomic DNA as a template, the DNA fragments composed of endogenous ppc and three Anderson promoters of different strengths (BBa_J23100, -107, -115; http:// parts. igem. org/) were amplified and then inserted into EcoRI site of pFT28-ectABC-EclysC* by One Step Cloning Kit, obtaining the plasmids pFT28-ectABC-EclysC*-ppc1, pFT28-ectABC-EclysC*-ppc2 and pFT28-ectABC-EclysC*-ppc3, respectively. In addition, the DNA fragment consisting of endogenous ppc and the promoter BBa_J23115 was also inserted into EcoRI site of pFT28-ectABC-EclysC*-aspDH by One Step Cloning Kit, resulting in the plasmid pFT28-ectABC-EclysC*-aspDH-ppc3.

Maps of all plasmids with the gene cluster ectABC con-structed in this study are shown in Fig. 2.

Strain construction

CRISPR-Cas9 system was utilized for deleting genes in the genome of E. coli strains according to the previously reported method [31]. To knock out crr, two homologous arms corresponding to the upstream and downstream regions of its locus were amplified using the primers crrF1, crrR1, crrF2 and crrR2. Then, the editing template fragment was obtained by overlap extension PCR using crrF1 and crrR2 as primers and the mixture of the two homologous arms as a template. In addition, the plasmid pTargetF-crr was extracted from the overnight culture using a TIANprep Mini Plasmid kit (Tiangen Biotech, Beijing, China), and it contained an N20 sequence for targeting the crr locus. Sub-sequently, the pTargetF-crr and the template fragment were simultaneously electroporated into MG1655, of which the

competent cells contained the lambda Red recombinase from pCas. Clones were selected on LB agar containing kanamy-cin (50 mg/L) and spectinomycin (50 mg/L) and successful crr knockout strains were confirmed by colony PCR using crrF1 and crrR2 as verified primers. Then pTargetF-crr was cured by adding 0.5 mM IPTG before curing pCas by incu-bation at 42 ℃ with shaking. MWZ001 was obtained after deleting crr from the genome of E. coli MG1655. The dele-tion of iclR, thrA and aspC was conducted in the same way as mentioned above, which resulted in the construction of MWZ002, MWZ003 and MWZ004, respectively (Table 1).

The ectoine-producing strains were constructed by elec-troporating the modified plasmids with the gene cluster ectABC and other endogenous or heterogeneous genes into the competent cells of corresponding hosts and verified by colony PCR using primers FW38-YF and FW38-YR.

Culture of ectoine‑producing strains in shake flasks and fed‑batch fermentation

All the medium used for culturing strains harboring vectors with ectoine synthesis encoding genes was supplemented with 0.9 mg/L triclosan for plasmid maintenance. The E. coli strains were first cultured on Luria–Bertani (LB) agar plates (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl and 15 g/L agar) for 24 h.

In shake flask fermentation, the bacterial pellets were scraped from the lawn of LB agar plates and cultivated in 50 mL sterilized LB medium (5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl) for 6 h at 37 °C and 200 rpm. This seed culture was used to inoculate 30 mL fermentation medium at an initial OD600 0.2 and incubated for 24 h or longer. The fermentation medium with an adjusted pH of 7.1 was modified based on the published medium formula [32], which contained 30 g/L glucose, 2 g/L yeast extract, 2 g/L citrate, 25 g/L (NH4)2SO4, 7.46 g/L KH2PO4, 2 g/L MgSO4·7 H2O, 5 mg/L FeSO4·7 H2O, 5 mg/L MnSO4·4 H2O, 0.8 mg/L Vitamin B1, 0.2 mg/L Vitamin H and 20 g/L CaCO3. At the beginning of fermentation, the incubation temperature was set at 37 °C for cell growth and then it was

Table 1 (continued) Strain/plasmid Description Source


Derived from pFT28-ectABC-EclysC*, inserting ppc under the control of the promoter PJ23115

This study


Derived from pFT28-ectABC-EclysC*, inserting This study

pFT28-ectABC-EclysC*-aspDH

aspDH under the control of the promoter PR This study

pFT28-ectABC-EclysC*-aspC

Derived from pFT28-ectABC-EclysC*, insertingaspC under the control of the promoter PR

This study

pFT28-ectABC-EclysC*-aspDH-ppc3

Derived from pFT28-ectABC-EclysC*-aspDH, inserting ppc with the control of the promoter PJ23115

http://parts.igem.org/


1 3

Table 2 Primers and artificial promoters used in this study

Names Sequence (5′–3′)

Primers crr-sgRNA-F ACC GTT GAA CTG AAA GGC GAG TTT TAG AGC TAG AAA TAGC crr-sgRNA-R TCG CCT TTC AGT TCA ACG GTA CTA GTA TTA TAC CTA GGA CTG AGC crrF1 TGC TGA AGG CAA ATG GAC crrR1 ATA ACA ACC GGA GTC AGG GTT CTT GTC GTC GGA AAC C crrF2 GGT TTC CGA CGA CAA GAA CCC TGA CTC CGG TTG TTA T crrR2 GGG ACT GGC GAC CTG TTT iclR-sgRNA-F ACG ATG AGG AAC ATG CAC TGG TTT TAG AGC TAG AAA TAGC iclR-sgRNA-R CAG TGC ATG TTC CTC ATC GTA CTA GTA TTA TAC CTA GGA CTG AGC iclRF1 CTT GTT GCT AAA GAT ATG ACG iclRR1 CAA ACC ATA CTG GCA TAA ACG CAG AGG CAA TAT TCT GCC CAT C iclRF2 GAT GGG CAG AAT ATT GCC TCT GCG TTT ATG CCA GTA TGG TTT G iclRR2 GAT CAG ATC CGC GCC ACC TTC thrA-sgRNA-F TGA TTG CGT AAT CAG CAC CAG TTT TAG AGC TAG AAA TAGC thrA-sgRNA-R TGG TGC TGA TTA CGC AAT CAA CTA GTA TTA TAC CTA GGA CTG AGC thrAF1 ATT ACC ACC ACC ATC ACC A thrAR1 CCA CTT CGG CAA TCT TCA CTT CAA TCA TCG CCA CCA G thrAF2 CTG GTG GCG ATG ATT GAA GTG AAG ATT GCC GAA GTG G thrAR2 CTG GCT GAT GAT GTC GTT TT aspC-sgRNA-F TGG TAA GCG AAG TCA AAC AGG TTT TAG AGC TAG AAA TAGC aspC-sgRNA-R CTG TTT GAC TTC GCT TAC CAA CTA GTA TTA TAC CTA GGA CTG AGC aspCF1 GTG GTT TAT GAT GCA CTG GGTT aspCR1 ACA ATC GCT TCG CAC AGC GGA GCG GCG GTA ATG TTC aspCF2 GAA CAT TAC CGC CGC TCC GCT GTG CGA AGC GAT TGT aspCR2 GGC TGA ACG AAG GCG ATA HR-ectABC-F TTA CCT CTT AAT TGG AGC TAGA GTT CAC ACA GGA AAC CTA CCAT ATG AAC GCA ACC ACA GAG CC ectABC-R CCA CTA GTT CTA GAG AGC TTT ACA GCG GCT TCT GGT CGTC lysC-cg1 GAT ATC ACT CGA GGTAC TAA CAC CGT GCG TGT TGA CTA TTT TAC CTC TGG CGG TGA TAA TGG TTG

CTTG GAG CTAGA GTT CAC ACA GGA AAC CTA CCATA TGG CCC TGG TCG TACA lysC-cg2 GAG GGC AGG TGA AGATGAT lysC-cg3 ATCA TCTT CAC CTG CCC TC lysC-cg4 AGA GTA ACA AAA GCT GAC TGC GAT GGT GGT CAT TGT lysCec1F GAT ATC ACT CGA GGTAC TAA CAC CGT GCG TGT TGA CTA TTT TAC CTC TGG CGG TGA TAA TGG TTG

CTCC GCT CTT CCC TTGTG lysCec1R TCA AGG ATTA ATG CCA CGC T lysCec2F AGC GTG GCA TTA ATCCT TGA lysCec2R TCT AGA GTA ACA AAA GCT GTT ACT CAA ACA AAT TAC TAT GCA PJ23100-ppc-F TTT TGC TGA AAG GAG TGG TTG ACG GCT AGC TCA GTC CTA GGT ACA GTG CTAGC GCA AAG CCC GAG

CAT ATT PJ23107-ppc-F TTT TGC TGA AAG GAG TGG TTT ACG GCT AGC TCA GCC CTA GGT ATT ATG CTAGC GCA AAG CCC GAG

CAT ATT PJ23115-ppc-F TTT TGC TGA AAG GAG TGG TTT ATA GCT AGC TCA GCC CTT GGT ACA ATG CTAGC GCA AAG CCC GAG

CAT ATT ppc-R TCG AGT GAT ATC GAA TTT TGC AGA AGA GGA AGA TTA GC aspC-F GTG GAA TTC GAT ATCAC TAA CAC CGT GCG TGT TGA CTA TTT TAC CTC TGG CGG TGA TAA TGG TTG

CGCG TTT TCA TCA GTA ATA GTTGG aspC-R CGG TGT TAG TAC CTC GAG CTT ACA GCA CTG CCA CAA T aspDH-F GAG TGG AAT TCG ATA TCA CTAA CAC CGT GCG TGT TGA CTA TTT TAC CTC TGG CGG TGA TAA TGG TTG

CCCT GCT GGA GGT GGA CAT aspDH-R CAC GGT GTT AGT ACC TCG AGG ATC GGG TTC AGG TCA AT FW38-YF TAC GAA CGG GGC GGA GAT T


1 3

The promoters are shown in italic; the RBS sequences are underlined, and the specific site directed mutation points are shown in bold

Table 2 (continued)

Names Sequence (5′–3′)

FW38-YR CGC TTG GAC TCC TGT TGA TAGA RT-16sRNA-F TCG GGA ACC GTG AGA CAG G RT-16sRNA-R CCG CTG GCA ACA AAG GAT AAG RT-ppc-F CCG TTT TAC TTC GTG GAT GG RT- ppc-R CAG CAG TTC AGG GGT CGC

Promoters PJ23100 (BBa_J23100) TTG ACG GCT AGC TCA GTC CTA GGT ACA GTG CTAGC PJ23107 (BBa_J23107) TTT ACG GCT AGC TCA GCC CTA GGT ATT ATG CTAGC PJ23115 (BBa_J23115) TTT ATA GCT AGC TCA GCC CTT GGT ACA ATG CTAGC

Fig. 2 Genetic comparison of constructed E. coli strains and maps of all plasmids with the gene cluster ectABC constructed in this study


1 3

increased to 42 °C for ectoine production 4 h or shorter/longer after the fermentation started. Samples at intervals of 6 h were taken to measure cell biomass and extracellular metabolites.

In fed-batch fermentation, the bacterial pellets were scraped and cultivated in 30 mL sterilized STF medium containing (NH4)2SO4 (15 g/L), sucrose (10 g/L), tryptone (20 g/L), yeast extract (5 g/L), and MgSO4 (1 g/L) for bet-ter cell growth [33]. After culturing for 6 h at 37 °C and 200 rpm, the seed culture was used to inoculate 100 mL STF medium at an initial OD600 0.2 and cultivated for 4 h at 37 °C and 200 rpm. Then, the culture was transferred into a 2.4-L bioreactor (T&J Minibox, China) with 700 mL fermentation medium, of which the formula was 40 g/L glucose, 2 g/L yeast extract, 4 g/L tryptone, 1 g/L sodium citrate tribasic dihydrate, 20 g/L (NH4)2SO4, 2 g/L KH2PO4, 0.7 g/L MgSO4, 100 mg/L FeSO4, 100 mg/L MnSO4, 0.8 mg/L Vitamin B1, 0.2 mg/L Vitamin H. The initial pH of the medium was adjusted to 7.0 by KOH, and maintained at 7.0 with NH3·H2O during fermentation. The temperature was controlled at 37 °C for 3 h at the beginning and turned up to 42 °C for the rest time of fermentation. The DO level (dissolved oxygen) was controlled above 20% by varying the aeration rate and the agitation speed (the maximum value was set at 900 rpm). The fermentation samples were taken every 3 h.

Analytical procedures

1 mL culture was taken at a time to monitor the growth and production of cells. The OD600 value was measured by a UV-1800 spectrophotometer (Shimadzu, Japan) to charac-terize cell growth. For measuring the level of the glucose remaining in the medium, an SBA-40C biosensor (Shan-dong, China) was utilized after centrifuging the taken culture 20 min at 13,800g and obtaining the supernatant.

The extracellular concentration of ectoine was directly measured after centrifuging the samples at 13,800g for 20 min and diluting the supernatants with pure water. The diluted samples were filtered through a 0.22-µm pore size membrane filter and then analyzed by isocratic HPLC (Agi-lent 1260 series, Hewlett-Packard) using a C18 column with acetonitrile/water mixture (70:30, v/v) at a flow rate of 0.8 mL/min as the mobile phase. UV detection at 210 nm was used to measure ectoine.

Relative transcription level of genes evaluated by real‑time quantitative PCR

The real-time quantitative PCR (RT-qPCR) was employed to evaluate relative transcription level of ppc under the control of promoters with different strengths, according to the previ-ously reported procedure [33]. Culturing different strains in

the same condition to the mid-exponential phase, the total RNA of each strain was extracted using RNA extraction kit (Bio Flux, Beijing, China). Then, the electrophoresis was used to check the amount and quality of total RNA.

After removing the residual DNA, the reverse transcrip-tion of cDNA was performed using HiScriptR II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). Then ABI Step One RT-PCR system (Applied Biosystems, San Mateo, CA, USA) and ChamQ™ Universal SYBRR qPCR Master Mix (Vazyme, Nanjing, China) were utilized to carry out RT-qPCR, of which the procedure began at 95 °C for 30 s, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. According to previously published method [34], the number of cycles required to get a fluorescent signal above the back-ground is called the cycle threshold value, based on which the relative abundance of the targeted mRNAs was quanti-fied. The relative abundance of 16S rRNA was used as an internal standard control to standardize the results.

Results and discussion

Increase ectoine production in E. coli by improving the accumulation of oxaloacetate

Due to its clear genetic background, E. coli K12 MG1655 was chosen as the original strain for de novo biosynthesis of many products [35]. MG1655 was also used in this study as the host for heterologous synthesis of ectoine. The high copy number plasmid pFT28-ectABC harboring the codon-optimized ectABC from H. elongate was introduced into MG1655. It is previously reported that the cell growth was apparently inhibited when ectoine was rapidly accumulated [20]. Therefore, we designed to control the expression of ect-ABC using a thermosensitive circuit cIts-pR-pL. The ectoine production was carried out by shake flask fermentation con-taining 30 mL medium. At the beginning of fermentation, the incubation temperature was set at 37 °C and the thermo-sensitive CI repressor bound the pR-pL promoter, inhibiting its transcription activity [36], so the cells did not express the gene cluster ectABC, leading to biomass accumulation. Then, the incubation temperature was increased to 42 °C, resulting in inactivation of CI repressor and activation of pR-pL promoter, and the ectABC gene cluster was function-ally expressed. The extracellular ectoine titer of MG1655/pFT28-ectABC reached 1.95 g/L after 36-h fermentation. The utilization of the thermosensitive circuit cIts-pR-pL to control the expression of ectABC makes ectoine produced without extra addition of any inducer, which has great poten-tial in large-scale industrial production.

As previously reported, the deletion of crr in E. coli remarkably increased l-threonine production [23], possi-bly because the absence of crr prevents the PEP-dependent


1 3

glucose uptake which consumes lots of PEP, the precur-sor for l-threonine biosynthesis (Fig. 1). Since the syn-thesis of l-threonine and ectoine needs the same precursor l-aspartate-β-semialdehyde (shown in Fig. 1), we speculated that deleting crr could also promote the biosynthesis of ectoine in E. coli. In addition, to further increase the precur-sor oxaloacetate, iclR was knocked out to reinforce the gly-oxylate shunt [27]. First, crr was deleted from MG1655 to get MWZ001, and then iclR was knocked out from MWZ001 to obtain MWZ002 (Table 1). As shown in Fig. 3, compared with MG1655/pFT28-ectABC, the extracellular ectoine titer of MWZ001/pFT28-ectABC improved 171% and reached to 5.28 g/L. In MWZ002/pFT28-ectABC, 9.09 g/L extracellular ectoine was achieved. These results indicate that directing carbon flux to oxaloacetate greatly improved the production of ectoine in E. coli.

Increase ectoine production in E. coli by reducing the carbon flux to competitive branch pathways and accumulating l‑aspartate‑β‑semialdehyde

Since ectoine and l-threonine share the precursor l-aspartate-β-semialdehyde (Fig. 1), the synthesis of ectoine should be enhanced if l-threonine synthesis pathway is weakened. Thus, thrA was deleted from MWZ002, resulting in MWZ003. However, the cell growth of MWZ003/pFT28-ectABC in the fermentation medium was severely inhibited and the glucose was seldom consumed, so the extracellular ectoine titer of MWZ003/pFT28-ectABC was too low to be measured (Fig. 3). This growth inhibition was not caused by the absence of l-threonine, because the cell growth was not recovered when extra l-threonine was supplemented in the fermentation medium (data not shown).

Another explanation for the growth inhibition of MWZ003/pFT28-ectABC could be that deleting thrA led to an insufficient level of aspartokinase and

Fig. 3 The effects of deleting crr, iclR and thrA as well as overex-pressing EclysC* or CglysC* on ectoine fermentation. a Cell growth; b Glucose consumption; c Ectoine titer. All experiments were per-formed in triplicate; error bars denote standard deviation of the mean.

The dashed lines refer to the temperature shifting time when the incu-bation temperature was increased from 37 to 42 °C inducing the syn-thesis of ectoine


1 3

l-aspartate-β-semialdehyde (Fig. 1). To supplement the defi-ciency of aspartokinase activity, two feedback-insensitive mutants, EclysC* (C1055T) encoding E. coli aspartokinase III [27] and CglysC* (G1A, C932T) encoding C. glutami-cum aspartokinase [37], were ligated into pFT28-ectABC and expressed under the control of PR, obtaining pFT28-ectABC-EclysC* and pFT28-ectABC-CglysC*, respectively. These two plasmids were first introduced to MWZ002 to test the effects of overexpressing EclysC* or CglysC* on ectoine production. As shown in Fig. 3, compared with MWZ002/pFT28-ectABC, the extracellular ectoine titer of MWZ002/pFT28-ectABC-EclysC* and MWZ002/pFT28-ectABC-CglysC* increased by 19% (i.e., 10.82 g/L) and 3% (i.e., 9.38 g/L), respectively. Subsequently, pFT28-ectABC-EclysC* and pFT28-ectABC-CglysC* were transferred into MWZ003, and their cells restored normal growth. Compared with MWZ002/pFT28-ectABC-EclysC*, the ectoine titer of MWZ003/pFT28-ectABC-EclysC* increased by 9.5% (i.e., 11.85 g/L). In comparison to MWZ002/pFT28-ectABC-CglysC*, the ectoine titer of MWZ003/pFT28-ectABC-CglysC* increased by 3% (i.e., 9.67 g/L). These results suggest that the aspartokinase from E. coli functioned bet-ter than that from C. glutamicum. In addition, after sup-plementing the aspartokinase activity, the deletion of thrA promoted the biosynthesis of ectoine. MWZ003/pFT28-ectABC-EclysC* with the highest ectoine titer was chosen for further study.

Modulate the overexpression of ppc to further promote ectoine synthesis in E. coli

The supply of oxaloacetate is one of the limiting factors for the synthesis of L-aspartate family amino acids [25] and an optimal level of PPC is required for efficient pro-duction of threonine [27], which should be accordant with the ectoine production. To further increase the accumula-tion of oxaloacetate, the ppc gene was additionally over-expressed in pFT28-ectABC-EclysC*. Three promoters (BBa_J23100, BBa_J23107, BBa_J23115) with different strengths were used to modulate the overexpression of ppc. The relative strengths of BBa_J23100, BBa_J23107 and BBa_J23115 were 1, 0.36 and 0.15, respectively, based on the measurement by Chris Anderson and the 2006 Berke-ley iGEM team (http:// parts. igem. org/). As shown in Fig. 4, MWZ003/pFT28-ectABC-EclysC*-ppc3 harboring the low-est strength promoter BBa_J23115 produced the highest titer of ectoine (11.98 g/L) after 24 h fermentation, while the ectoine titer in MWZ003/pFT28-ectABC-EclysC*-ppc1 and MWZ003/pFT28-ectABC-EclysC*-ppc2 reached 7.93 g/L and 10.07 g/L, respectively. These results indicate that the strengths of promoters BBa_J23100 and BBa_J23107 were too strong while the lowest strength promoter BBa_J23115

was the best for the overexpression of ppc to ectoine biosyn-thesis. Using MWZ003/pFT28-ectABC-EclysC* as the con-trol, the transcriptional level of the gene ppc in MWZ003/pFT28-ectABC-EclysC*-ppc1, MWZ003/pFT28-ectABC-EclysC*-ppc2, and MWZ003/pFT28-ectABC-EclysC*-ppc3 were evaluated by RT-qPCR (Fig. 4d). As expected, the rela-tive transcription level of ppc was accordant with relative strength of the three promoters. The results suggest that moderate overexpression of ppc promoted ectoine biosyn-thesis, but excessive PPC activity had an adverse influence.

Generally, ppc overexpression increases oxaloacetate which in turn contributes to ectoine production. However, when too much PEP is converted to oxaloacetate through ppc overexpression, there will be the flux imbalance between oxaloacetate and acetyl-coA, which is converted from PEP as well. This is not beneficial for the synthesis of ectoine, because equimolar oxaloacetate and acetyl-coA are required as precursors for ectoine synthesis. Therefore, there should be an optimal level of ppc expression for ectoine production in MWZ003/pFT28-ectABC-EclysC*. The overexpression level of ppc controlled by the weakest promoter BBa_J23115 is closer to the optimal level than the other two promoters, thus leading to the highest ectoine production.

Introduce a novel aspartate dehydrogenase from P. aeruginosa PAO1 to promote ectoine synthesis in E. coli

Since ectoine is an aspartic acid-derived chemical, the aspartate aminotransferase (AspC)-catalyzed reaction was enhanced to improve the synthesis of ectoine by further over-expressing aspC. However, the ectoine titer of MWZ003/pFT28-ectABC-EclysC*-aspC (11.69 g/L) was only slightly higher than that of MWZ003/pFT28-ectABC-EclysC* (11.32 g/L) (Fig. 4). To verify the expression effectiveness of aspC on the plasmid, the chromosomal aspC was deleted in MWZ003, obtaining MWZ004. When pFT28-ectABC-EclysC*-aspC was transferred into MWZ004, the resulting strain MWZ004/pFT28-ectABC-EclysC*-aspC produced almost the same amount of ectoine as MWZ003/pFT28-ectABC-EclysC* (data not shown). Therefore, overexpres-sion of aspC did not significantly increase the production of ectoine, which was consistent with previously reported studies on other aspartic acid-derived products [25, 26].

The AspC-catalyzed reaction uses oxaloacetate and glu-tamate to generate aspartic acid, while glutamate is also consumed by the conversion of l-aspartate-β-semialdehyde to l-2,4-diaminobutyrate. These two reactions compete for the glutamate as the substrate, which may limit the effect of overexpressing aspC. A aspartate dehydrogenase from P. aeruginosa PAO1 (PaeAspDH) aminates OAA directly to form aspartic acid using both nicotinamide adenine

http://parts.igem.org/


1 3

dinucleotide (NADH) and nicotinamide adenine dinucleo-tide phosphate (NADPH) as coenzyme, therefore, it can be applied to ease the tight supply of glutamate and add an extra pathway to convert oxaloacetate to aspartic acid. The genes aspDH encoding aspartate dehydrogenase was cloned from the genome of P. aeruginosa PAO1 and inserted into pFT28-ectABC-EclysC*, obtaining pFT28-ectABC-EclysC*-aspDH. As shown in Fig. 4, the ectoine titer of MWZ003/pFT28-ectABC-EclysC*-aspDH (12.27 g/L) improved by 8.39% compared with that of MWZ003/pFT28-ectABC-EclysC* (11.32 g/L). The plasmid pFT28-ectABC-EclysC*-aspDH was also introduced into MWZ004 to test if the activity of PaeAspDH could completely replace that of AspC. The ectoine titer of MWZ004/pFT28-ectABC-EclysC*-aspDH

(3.00 g/L) decreased dramatically in comparison to that of MWZ003/pFT28-ectABC-EclysC*-aspDH.

Optimize shake flask fermentation conditions of MWZ003/pFT28‑ectABC‑EclysC*‑aspDH‑ppc3 to improve ectoine production

From the results described above, the overexpression of ppc and aspDH under the control of the promoter BBa_J23115 and PR, respectively, could improve the produc-tion of ectoine, so the two manipulations were combined to construct the plasmid pFT28-ectABC-EclysC*-aspDH-ppc3, which was transferred into MWZ003 to form the final strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3. The ectoine titer reached 12.93 g/L after 24 h fer-mentation with the temperature shifting from 37 to 42 ℃ 4 h after the fermentation started, higher than that

Fig. 4 The effects of ppc overexpression modulated by varying gradi-ent strength promoters and overexpressing aspDH or aspC on ectoine fermentation. a Cell growth; b Glucose consumption; c Ectoine titer. d Relative transcription levels of ppc in MWZ003/pFT28-ectABC-EclysC*, MWZ003/pFT28-ectABC-EclysC*-ppc1, MWZ003/pFT28-

ectABC-EclysC*-ppc2 and MWZ003/pFT28-ectABC-EclysC*-ppc3 analyzed by RT-qPCR. All experiments were performed in triplicate; error bars denote standard deviation of the mean. The dashed lines refer to temperature shifting time when the incubation temperature was increased from 37 to 42 °C inducing the synthesis of ectoine


1 3

MWZ003/pFT28-ectABC-EclysC*-ppc3 or MWZ003/pFT28-ectABC-EclysC*-aspDH.

The switching time of the thermal switch system is important for enhancing target metabolite production [32]. Therefore, the time points for temperature shifting were set at 0, 2, 3, 4, 5, or 6 h after the start of fermentation, and the cell growth, glucose consumption, and ectoine production during fermentation of MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 were investigated. As shown in Fig. 5a, the final cell density increased with the postponing of tempera-ture shift. However, the increase of biomass accumulation showed a negative correlation with ectoine productivity when the amount of available glucose was constant. The final ectoine titer (14.25 g/L) reached the highest level when the temperature was changed at 3 h after the fermentation began. These results suggest that the selection of the proper

time point for temperature changing could further improve the ectoine production efficiency in E. coli. The temperature shifting time of the following shake flask fermentation was set at 3 h after the fermentation started.

According to a previous study, deletion of the gene crr in E. coli could improve the tolerance to glucose [23]. To evaluate its production performance, the fermentation of the engineered strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 was carried out in shake flasks containing the fermentation medium with different initial glucose concen-trations, shifting the temperature from 37 to 42 ℃ at 3 h after the fermentation began. As shown in Fig. 5b, the final cell density increased with the enhancement of initial glu-cose concentrations, while the glucose consumption rate had no significant difference and the glucose was used out 36 h after fermentation started (Fig. 5c). The results of ectoine

Fig. 5 Optimization of fermentation conditions for ectoine produc-tion in shake flask scale. a The effect of different temperature shift-ing time on the ectoine production of the final engineered strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3. The biomass (OD600) and ectoine titer after 24-h fermentation are shown. The glu-cose in fermentation media was used up at 24 h, of which the initial concentration was 30 g/L. Temperature shifting time refers to how many hours after the start of fermentation when the temperature was raised from 37 to 42 ℃. b Cell growth of the final engineered strain

with different initial glucose concentrations. c Glucose consumption of the final engineered strain with different initial glucose concentra-tions. d Production performance of the final engineered strain with different initial glucose concentrations. The dashed lines refer to tem-perature shifting time when the incubation temperature was increased from 37 to 42 °C inducing the synthesis of ectoine. All experiments were performed in triplicate; error bars denote standard deviation of the mean


1 3

production performance are shown in Fig. 5d. Cultured in the medium with 40 g/L initial glucose, the ectoine titer (17.89 g/L) reached the highest level, which improved by 26.16% in contrast to that when cultured in the medium with 30 g/L initial glucose. When the initial glucose concentra-tion was higher than 40 g/L, the ectoine titer decreased with the increase of glucose concentration, which may be caused by glucose effect [38].

Ectoine production performance of the final strain by fed‑batch fermentation

Using MG1655/pFT28-ectABC as the control, the ectoine production of the final strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 was investigated in the scale of fed-batch fermentation (Fig. 6). The final cell density of MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 was much higher than that of the control. The initial glucose concentration was 40 g/L, and extra glucose was supple-mented to maintain the glucose concentration between 10 and 25 g/L. After 36-h fermentation, MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 consumed 184 g glucose in total and the ectoine titer reached 30.37 g/L. By contrast, MG1655/pFT28-ectABC consumed 136.8 g glucose totally and produced 10.51 g/L ectoine. The overall ectoine yield from glucose of the final strain and the control strain were 0.132 g/g and 0.06 g/g, respectively.

Conclusion

In this study, a rationally engineered E. coli strain which could produce ectoine efficiently without the addition of inducers or the control of osmolality was constructed.

This was accomplished by (1) constructing the ectoine synthesis pathway in E. coli MG1655 by the introduc-tion of codon-optimized ectABC gene cluster from H. elongata; (2) enhancing the supply of oxaloacetate by deleting crr and iclR; (3) weakening the competitive pathways by deleting thrA and supplementing the activ-ity of aspartokinase by overexpressing an endogenous feedback-resistant lysC gene; (4) moderating overexpres-sion of ppc to promote ectoine synthesis; (5) launching an extra pathway to convert oxaloacetate to aspartate by recruiting heterologous aspartate dehydrogenase derived from P. aeruginosa PAO1. Fed-batch fermentation of the final engineered strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 achieved a production of 30.37 g/L ectoine with glucose as the carbon source, and the yield and productivity reached 0.132 g/g glucose and 0.84 g/(L h), respectively.

Author contributions SZ, YF and XW conceived and designed the research. SZ, YF, LZ, HL, ZW, and YL conducted the experiments. SZ, YF and XW analyzed the data and wrote the manuscript. All the authors read and approved the manuscript.

Funding This study was supported by the National Key R&D Program of China (2018YFA0900300), the National First-class Discipline Pro-gram of Light Industry Technology and Engineering (LITE2018-10), the Key Technology Project of Inner Mongolia Autonomous Region in China (2019GG302), and the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation.

Availability of data and material The datasets generated and/or ana-lyzed during the current study are available from the corresponding author on reasonable request. In addition, the GeneBank accession number of the 2433-bp codon-optimized ectABC gene cluster is MW316739.

Code availability Not applicable.

Fig. 6 Fed-batch fermentation for ectoine production in MG1655/pFT28-ectABC (a) and MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 (b)


1 3

Declarations

Conflict of interest On behalf of all the authors, the corresponding au-thor states that there is no conflict of interest.

Compliance with ethics requirements This article does not contain any studies with human participants or animals performed by any of the authors.

References

1. Galinski EA, Pfeiffer HP, Trüper HG. 1,4,5,6-Tetrahydro-2-me-thyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur J Biochem. 1985;149:135–9.

2. Lentzen G, Schwarz T. Extremolytes: natural compounds from extremophiles for versatile applications. Appl Microbiol Biotech-nol. 2006;72:623–34.

3. Graf R, Anzali S, Buenger J, Pfluecker F, Driller H. The multi-functional role of ectoine as a natural cell protectant. Clin Der-matol. 2008;26:326–33.

4. Kanapathipillai M, Lentzen G, Sierks M, Park CB. Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzhei-mer’s beta-amyloid. FEBS Lett. 2005;579:4775–80.

5. Sydlik U, Weissenberg A, Peuschel H, Krutmann J, Unfried K. The compatible solute ectoine protects against nanopar-ticle-induced neutrophilic lung inflammation. Toxicol Lett. 2009;189:S188–S188.

6. Abdel-Aziz H, Wadie W, Abdallah DM, Lentzen G, Khayyal MT. Novel effects of ectoine, a bacteria-derived natural tetrahydropy-rimidine, in experimental colitis. Phytomedicine. 2013;20:585–91.

7. Czech L, Hermann L, Stöveken N, Richter AA, Höppner A, Smits SHJ, Heider J, Bremer E. Role of the extremolytes ectoine and hydroxyectoine as stress protectants and nutrients: genetics, phylogenomics, biochemistry, and structural analysis. Genes. 2018;9:177.

8. Pastor JM, Salvador M, Argandona M, Bernal V, Reina-Bueno M, Csonka LN, Iborra JL, Vargas C, Nieto JJ, Canovas M. Ectoines in cell stress protection: uses and biotechnological production. Biotechnol Adv. 2010;28:782–801.

9. Sauer T, Galinski EA. Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol Bioeng. 1998;57:306–13.

10. Fallet C, Rohe P, Franco-Lara E. Process optimization of the integrated synthesis and secretion of ectoine and hydroxyec-toine under hyper/hypo-osmotic stress. Biotechnol Bioeng. 2010;107:124–33.

11. Schubert T, Maskow T, Benndorf D, Harms H, Breuer U. Con-tinuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium. Appl Environ Micro-biol. 2007;73:3343–7.

12. Grammann K, Volke A, Kunte HJ. New type of osmoregu-lated solute transporter identified in halophilic members of the Bacteria domain: TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581(T). J Bacteriol. 2002;184:3078–85.

13. Tetsch L, Kunte HJ. The substrate-binding protein TeaA of the osmoregulated ectoine transporter TeaABC from Halomonas elongata: purification and characterization of recombinant TeaA. FEMS Microbiol Lett. 2002;211:213–8.

14. Zhang L-H, Lang Y-J, Nagata S. Efficient production of ectoine using ectoine-excreting strain. Extremophiles. 2009;13:717–24.

15. Pérez-García F, Ziert C, Risse JM, Wendisch VF. Improved fermentative production of the compatible solute ectoine by Corynebacterium glutamicum from glucose and alternative carbon sources. J Biotechnol. 2017;258:59–68.

16. Becker J, Schäfer R, Kohlstedt M, Harder BJ, Borchert NS, Stöveken N, Bremer E, Wittmann C. Systems metabolic engineering of Corynebacterium glutamicum for produc-tion of the chemical chaperone ectoine. Microb Cell Fact. 2013;12:110–110.

17. Giesselmann G, Dietrich D, Jungmann L, Kohlstedt M, Jeon E, Yim SS, Sommer F, Zimmer D, Mühlhaus T, Schroda M, Jeong K, Becker J, Wittmann C. Metabolic engineering of Corynebac-terium glutamicum for high-level ectoine production: design, combinatorial assembly, and implementation of a transcrip-tionally balanced heterologous ectoine pathway. Biotechnol J. 2019;14:e201800417.

18. He Y-Z, Gong J, Yu H-Y, Tao Y, Zhang S, Dong Z-Y. High pro-duction of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli. Microb Cell Fact. 2015;14:55.

19. Chen W, Zhang S, Jiang P, Yao J, He Y, Chen L, Gui X, Dong Z, Tang S-Y. Design of an ectoine-responsive AraC mutant and its application in metabolic engineering of ectoine biosynthesis. Metab Eng. 2015;30:149–55.

20. Ning Y, Wu X, Zhang C, Xu Q, Chen N, Xie X. Pathway con-struction and metabolic engineering for fermentative production of ectoine in Escherichia coli. Metab Eng. 2016;36:10–8.

21. Ma H, Zhao Y, Huang W, Zhang L, Wu F, Ye J, Chen G-Q. Rational flux-tuning of Halomonas bluephagenesis for co-production of bioplastic PHB and ectoine. Nat Commun. 2020;11:3313–3313.

22. Plumbridge J. Regulation of gene expression in the PTS in Escher-ichia coli: the role and interactions of Mlc. Curr Opin Microbiol. 2002;5:187–93.

23. Zhu LF, Fang Y, Ding ZX, Zhang SY, Wang XY. Developing an l-threonine-producing strain from wild-type Escherichia coli by modifying the glucose uptake, glyoxylate shunt, and l-threonine biosynthetic pathway. Biotechnol Appl Biochem. 2019;15:962–76.

24. Zhao H, Fang Y, Wang XY, Zhao L, Wang JL, Li Y. Increasing l-threonine production in Escherichia coli by engineering the glyoxylate shunt and the l-threonine biosynthesis pathway. Appl Microbiol Biotechnol. 2018;102:5505–18.

25. Song CW, Lee J, Ko Y-S, Lee SY. Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid. Metab Eng. 2015;30:121–9.

26. Zou X, Guo L, Huang L, Li M, Zhang S, Yang A, Zhang Y, Zhu L, Zhang H, Zhang J, Feng Z. Pathway construction and metabolic engineering for fermentative production of β-alanine in Escheri-chia coli. Appl Microbiol Biotechnol. 2020;104:2545–59.

27. Lee KH, Park JH, Kim TY, Kim HU, Lee SY. Systems metabolic engineering of Escherichia coli for Kl-threonine production. Mol Syst Biol. 2007;3:149–149.

28. Li Y, Kawakami N, Ogola HJO, Ashida H, Ishikawa T, Shibata H, Sawa Y. A novel l-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characteri-zation and application for l-aspartate production. Appl Microbiol Biotechnol. 2011;90:1953–62.

29. Li YJ, Wei HB, Wang T, Xu QY, Zhang CL, Fan XG, Ma Q, Chen N, Xie XX. Current status on metabolic engineering for the production of l-aspartate family amino acids and derivatives. Bioresour Technol. 2017;245:1588–602.

30. Wendisch VF. Metabolic engineering advances and prospects for amino acid production. Metab Eng. 2020;58:17–34.


1 3

31. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene edit-ing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015;81:2506–14.

32. Fang Y, Wang J, Ma W, Yang J, Zhang H, Zhao L, Chen S, Zhang S, Hu X, Li Y, Wang X. Rebalancing microbial carbon distribu-tion for l-threonine maximization using a thermal switch system. Metab Eng. 2020;61:33–46.

33. Yang J, Fang Y, Wang J, Wang C, Zhao L, Wang X. Deletion of regulator-encoding genes fadR, fabR and iclR to increase l-thre-onine production in Escherichia coli. Appl Microbiol Biotechnol. 2019;103:4549–64.

34. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8.

35. Zhang W, Liu Z, Gong M, Li N, Lv X, Dong X, Liu Y, Li J, Du G, Liu L. Metabolic engineering of Escherichia coli for the

production of lacto-N-neotetraose (LNnT). Syst Microbiol Bio-manuf. 2021. https:// doi. org/ 10. 1007/ s43393- 021- 00023-1.

36. Sugino Y, Morita M. Cold-sensitive phenotype of Escherichia coli cells harboring a plasmid carrying the kil gene of phage lambda brought under control of cI857 gene promoters. Gene. 1994;141:25–30.

37. Becker J, Zelder O, Häfner S, Schröder H, Wittmann C. From zero to hero—design-based systems metabolic engineering of Corynebacterium glutamicum for l-lysine production. Metab Eng. 2011;13:159–68.

38. Rédei GP. Glucose effect. In: Rédei GP, editor. Encyclopedia of genetics, genomics, proteomics and informatics. Dordrecht: Springer; 2008. p. 803–4.

https://doi.org/10.1007/s43393-021-00023-1

Documents

Metabolic engineering of Escherichia coli for efficient