High‐Level Gene Expression for Recombinant Penicillin Acylase Production Using the <i>araB</i> Promoter System in <i>Escherichia coli</i>

Narayanan, Niju; Xu, Yunfei; Chou, C. Perry

doi:10.1021/bp060135u

Cited by 11 publications

(7 citation statements)

References 40 publications

(43 reference statements)

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“…For enzymatic reporter assays, β-D-glucuronidase (UidA) was used (Kallnik et al 2010). UidA activity was determined in a Miller assay essentially as described (Miller 1992). Immediately after inoculation of G. oxydans shake flask cultures, uidA expression was induced by the addition of 1% (w/ v) L-arabinose.…”

Section: Measurements Of Fluorescence Protein and Enzyme Activitymentioning

confidence: 99%

A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans

Fricke

Link

Gätgens

et al. 2020

Appl Microbiol Biotechnol

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The acetic acid bacterium (AAB) Gluconobacter oxydans incompletely oxidizes a wide variety of carbohydrates and is therefore used industrially for oxidative biotransformations. For G. oxydans, no system was available that allows regulatable plasmid-based expression. We found that the l-arabinose-inducible PBAD promoter and the transcriptional regulator AraC from Escherichia coli MC4100 performed very well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporters β-glucuronidase and mNeonGreen, up to 480-fold induction with 1% l-arabinose, and tunability from 0.1 to 1% l-arabinose. In G. oxydans 621H, l-arabinose was oxidized by the membrane-bound glucose dehydrogenase, which is absent in the multi-deletion strain BP.6. Nevertheless, AraC-PBAD performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-PBAD in wild-type G. oxydans strains. However, the oxidation product arabinonic acid strongly contributed to the acidification of the growth medium in 621H cultures during the stationary phase, which resulted in drastically decreased reporter activities in 621H (pH 3.3) but not in BP.6 cultures (pH 4.4). These activities could be strongly increased quickly solely by incubating stationary cells in d-mannitol-free medium adjusted to pH 6, indicating that the reporters were hardly degraded yet rather became inactive. In a pH-controlled bioreactor, these reporter activities remained high in the stationary phase (pH 6). Finally, we created a multiple cloning vector with araC-PBAD based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-PBAD system in G. oxydans that could possibly also be used in other AAB. Key points • We found the AraC-PBADsystem from E. coli MC4100 was well tunable in G. oxydans. • In the absence of AraC orl-arabinose, expression from PBADwas extremely low. • This araC-PBADsystem could also be fully functional in other acetic acid bacteria.

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Section: Measurements Of Fluorescence Protein and Enzyme Activitymentioning

confidence: 99%

A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans

Fricke

Link

Gätgens

et al. 2020

Appl Microbiol Biotechnol

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“…This growth decoupled expression system is based on E. coli strain BL21(DE3), which includes a chromosomal copy of T7 RNAP controlled by the IPTG inducible P lacUV5 and additional has a deletion of araBAD gene cluster to avoid metabolization of l-arabinose. Thereby, we can rule out that induction of lac-promoters is due to an araBAD-derived l-arabinose degradation product (as suggested by Narayanan et al) [23,25]. Instead, it seems that l-arabinose can indeed bind or interact with LacI and thereby allows transcription of P lacUV5 controlled T7 RNAP, and in the same way derepressing the P T7 controlled GOI located on the pET-based plasmid.…”

Section: Resultsmentioning

confidence: 80%

“…It was already shown in previous studies that l-arabinose can induce lac-derived P trc or P lacUV5 /P T7-lacO promoter in the E. coli strains JM109/JM109(DE3) and thereby allowing better soluble expression and less inclusion body formation of Penicillin G acylase (PAC) [22][23][24]. Further, it was shown that expression of PAC in E. coli strains MD∆P7 (mutation in araC) and MC4100 (mutation in araD) failed in showing equal PAC expression results from lac-derived P trc with the induction of l-arabinose compared to JM109, hypothesizing that the derived metabolites of l-arabinose are responsible for the binding of the LacI repressor and that induction is not mediated by l-arabinose directly [23,25]. In E. coli l-arabinose is converted into l-ribulose (araA), l-ribulose-5-phosphate (araB), and finally d-xylulose-5-phosphate (araD), subsequently drained into the pentose phosphate pathway.…”

Section: Introductionmentioning

confidence: 99%

Tunable expression rate control of a growth-decoupled T7 expression system by l-arabinose only

Stargardt¹,

Striedner

Mairhofer³

2021

Microb Cell Fact

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Background Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with l-arabinose in a newly constructed Escherichia coli expression host BL21-AI<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. Results Here, we show that BL21-AI<gp2> is able to precisely regulate protein production rates on a cellular level in an l-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting. Conclusions In all cases tested, BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.

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“…The pET expression system has a pelB secretion signal at the N-terminus, which directs the results of polypeptide synthesis to the periplasm of E. coli [16]. Host E. coli HB101 is also used for target protein expression because this strain excels in periplasmic recombinant protein expression [2,17].…”

Section: Expression Of Pga Recombinant Protein and Enzyme Activity Testmentioning

confidence: 99%

Expression of Synthetic pac Gene Encoding Penicillin G Acylase (PGA) Enzyme in E. coli BL21(DE3) and HB101

Amin,

Sismindari,

Purwanto

2024

Curr. Appl. Sci. Technol.

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High bacterial infection cases in Indonesia cause a high need for antibiotic drugs. Unfortunately, most of the raw materials used for antibiotic production in Indonesia are still imported. For this reason, the government is eager to find better ways to produce penicillin and its derivatives, which are widely used in society. The production of penicillin-derivative requires penicillin G acylase (PGA) as a catalyst. In previous studies, the expression of the syn-pac gene in E. coli BL21(DE3) to produce a recombinant PGA enzyme was performed, but the enzyme activity was low (0.01754 U/mg). Thus, the expression was carried out in different hosts and inducers. The purpose of this research was to obtain the production of PGA with higher enzyme activity. The transformation was carried out in the pET22b-pacEc in E. coli BL21(DE3) and HB101. For enzyme expression, the recombinant hosts were induced by 0.05 mM IPTG, 176 mM lactose, and 1998 mM arabinose at a temperature of 20°C and 150 rpm of shaking for 17 h. Protein isolation was performed by sonication and freeze-thawing to recover biologically active PGA. Verification of PGA was performed by SDS-PAGE and the enzyme activity was tested by pDAB. E. coli HB101 produced PGA with higher activity (10.17 U/mg) than BL21(DE3) (6.67 U/mg), and arabinose was the strongest inducer for enzyme expression.

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High‐Level Gene Expression for Recombinant Penicillin Acylase Production Using the araB Promoter System in Escherichia coli

Cited by 11 publications

References 40 publications

A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans

A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans

Tunable expression rate control of a growth-decoupled T7 expression system by l-arabinose only

Expression of Synthetic pac Gene Encoding Penicillin G Acylase (PGA) Enzyme in E. coli BL21(DE3) and HB101

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