Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

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A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression inKluyveromyces lactis

Applied and Environmental Microbiology ◽

10.1128/aem.00542-19 ◽

2019 ◽

Vol 85 (14) ◽

Cited By ~ 2

Author(s):

Hassan Sakhtah ◽

Juliane Behler ◽

Alana Ali-Reynolds ◽

Thomas B. Causey ◽

Saulius Vainauskas ◽

...

Keyword(s):

Gene Expression ◽

Carbon Source ◽

Protein Expression ◽

Recombinant Proteins ◽

Heterologous Protein ◽

Kluyveromyces Lactis ◽

Heterologous Protein Expression ◽

Heterologous Proteins ◽

Content Type ◽

Hybrid Promoter

ABSTRACTThe yeastKluyveromyces lactishas been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressibleLAC4promoter (PLAC4) is the most widely used promoter to drive recombinant protein expression inK. lactis. However, PLAC4is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, PLAC4is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novelK. lactispromoter system that is both strong and tightly controllable. We first tested several different endogenousK. lactispromoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P350) was created by combining segments of twoK. lactispromoters, namely, the strong constitutive PGAP1promoter and the carbon source-sensitive PICL1promoter. We demonstrate that P350is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P350-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P350hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins inK. lactis.IMPORTANCEThe yeastKluyveromyces lactisis an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P350) for use inK. lactis. P350is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be “autoinduced” at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.

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Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know

Current Pharmaceutical Design ◽

10.2174/1381612824666180131121940 ◽

2018 ◽

Vol 24 (6) ◽

pp. 718-725 ◽

Cited By ~ 7

Author(s):

Seyed Mohammad Gheibi Hayat ◽

Najmeh Farahani ◽

Behrouz Golichenari ◽

Amirhossein Sahebkar

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Proteins ◽

Recombinant Protein Expression ◽

Culture Conditions ◽

Strain Vector ◽

Factors Affecting ◽

Expression In Escherichia Coli ◽

Pros And Cons

Background: Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. Methodology: A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Results: Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Conclusion: Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer.

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Transformation of, and Heterologous Protein Expression in,Lactobacillus agilisandLactobacillus vaginalisIsolates from the Chicken Gastrointestinal Tract

Applied and Environmental Microbiology ◽

10.1128/aem.02006-10 ◽

2010 ◽

Vol 77 (1) ◽

pp. 220-228 ◽

Cited By ~ 9

Author(s):

David P. Stephenson ◽

Robert J. Moore ◽

Gwen E. Allison

Keyword(s):

Gastrointestinal Tract ◽

Protein Expression ◽

Reporter Gene ◽

Heterologous Protein ◽

High Protein Diet ◽

Plasmid Vector ◽

Heterologous Protein Expression ◽

Heterologous Proteins

ABSTRACTLactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derivedLactobacillusstrains,Lactobacillus agilisLa3,Lactobacillus vaginalisLv5, andLactobacillus crispatusLc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 108and 106transformants per microgram of plasmid DNA, respectively. The third strain tested,L. crispatusLc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter genegfpwas analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3ldhLpromoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression inL. agilisandL. vaginalis. The ability of these strains to express heterologous proteinsin vitroindicates their potential utility asin vivodelivery vectors for therapeutic peptides to the chicken gastrointestinal tract.

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Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

Frontiers in Microbiology ◽

10.3389/fmicb.2021.682001 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gema Lozano Terol ◽

Julia Gallego-Jara ◽

Rosa Alba Sola Martínez ◽

Adrián Martínez Vivancos ◽

Manuel Cánovas Díaz ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Proteins ◽

Copy Number ◽

Recombinant Protein Production ◽

Protein Production ◽

Recombinant Protein Expression ◽

Expression System ◽

E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

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Multimodal Approaches for the Improvement of the Cellular Folding of a Recombinant Iron Regulatory Protein in E. Coli

10.21203/rs.3.rs-847274/v1 ◽

2021 ◽

Author(s):

Gayathri Ravitchandirane ◽

Sheetal Bandhu ◽

Tapan K. Chaudhuri

Keyword(s):

Heat Shock ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Proteins ◽

Physiological Stress ◽

Recombinant Protein Expression ◽

Host Cells ◽

Growth Media ◽

E Coli ◽

Expression Host

Abstract BackgroundDuring the recombinant protein expression, foreign proteins are generated in insoluble and inactive aggregates in E. coli cell factories, which inhibits E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The process variables such as incubation temperature, inducer concentrations, growth media, and the presence of folding modulators such as exogenous molecular chaperones or osmolytes are crucial for the cellular folding and are investigated in the study. The processes the physiological stress such as osmotic and heat shock stimulation in the host cells and thereby their effect on the solubility and activity of recombinant proteins was also analysed.ResultsOf the various methods discussed, the cells subjected to the addition of osmolytes and pre-induction heat shock exhibited significant enhancement in the recombinant aconitase activity. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells.ConclusionsThe recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase productivity during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression.

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A Comprehensive Guide to the Commercial Baculovirus Expression Vector Systems for Recombinant Protein Production

Protein and Peptide Letters ◽

10.2174/0929866526666191112152646 ◽

2020 ◽

Vol 27 (6) ◽

pp. 529-537 ◽

Cited By ~ 1

Author(s):

Vibhor Mishra

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Expression Vector ◽

Recombinant Protein Expression ◽

Scale Up ◽

Vector System ◽

Heterologous Protein Expression ◽

Baculovirus Expression ◽

Vector Systems ◽

Baculovirus Expression Vector

The Baculovirus Expression Vector System (BEVS) is a workhorse for recombinant protein expression for over thirty-five years. Ever since it was first used to overexpress the human IFN-β protein, the system has been engineered and modified several times for quick and easy expression and scale-up of the recombinant proteins. Multiple gene assemblies performed on the baculovirus genome using synthetic biology methods lead to optimized overexpression of the multiprotein complexes. Nowadays, several commercially available BEVS platforms offer a variety of customizable features, and often it is confusing which one to choose for a novice user. This short review is intended to be a one-stop guide to the commercially available baculovirus technology for heterologous protein expression in the insect cells, which users can refer to choose from popular and desirable BEVS products or services.

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Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii

Microbiology ◽

10.1099/mic.0.030064-0 ◽

2009 ◽

Vol 155 (11) ◽

pp. 3581-3588 ◽

Cited By ~ 9

Author(s):

Song F. Lee ◽

Yi-Jing Li ◽

Scott A. Halperin

Keyword(s):

Protein Expression ◽

Codon Usage ◽

Codon Usage Bias ◽

Heterologous Protein ◽

Trna Gene ◽

Heterologous Protein Expression ◽

Streptococcus Gordonii ◽

Heterologous Proteins ◽

Single Chain ◽

Rare Codons

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

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Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

International Journal of Molecular Sciences ◽

10.3390/ijms21030928 ◽

2020 ◽

Vol 21 (3) ◽

pp. 928 ◽

Cited By ~ 2

Author(s):

Sviatlana Smolskaya ◽

Yulia A. Logashina ◽

Yaroslav A. Andreev

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Dna ◽

Recombinant Protein Expression ◽

Expression System ◽

Reaction Medium ◽

Cellular Metabolism ◽

Heterologous Protein Expression

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

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The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli

Frontiers in Microbiology ◽

10.3389/fmicb.2021.779541 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lei Zou ◽

Sha Li ◽

Nan Li ◽

Shi-Long Ruan ◽

Jing Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Expression ◽

Heterologous Protein ◽

Fluorescent Protein ◽

Recombinant Protein Expression ◽

Yellow Fluorescent Protein ◽

Fluorescence Emission ◽

Soluble Expression ◽

Heterologous Protein Expression ◽

E Coli

Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.

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Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains

Nepal Journal of Biotechnology ◽

10.3126/njb.v2i1.5634 ◽

1970 ◽

Vol 2 (1) ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Santosh Dulal ◽

Bhupal Ban ◽

Gi Hyoek Yang ◽

Hyun Ho Jung

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Recombinant Proteins ◽

Clostridium Botulinum ◽

Pathogen Detection ◽

Recombinant Protein Expression ◽

Synthetic Genes ◽

Effective System ◽

Genes Encoding

The use of recombinant BoNT domains has been proposed as a means to develop strategies to treat and prevent botulism. Here, details on the molecular cloning, protein expression, purification, and immunoreactivity of BoNT/F domains from Clostridium botulinum are presented. Initially, full-length synthetic genes encoding recombinant BoNT/F domains (catalytic, translocation, and receptor binding) were designed and cloned into Escherichia coli for expression. Recombinant proteins were then purified through GST affinity chromatography preceding elution of GST-free recombinant domains by thrombin protease. Soluble recombinant proteins encoding catalytic light chain and translocation N-terminal heavy chain were subsequently used to perform in vivo immunization. Polyclonal mouse antibodies specific to these domains were raised, confirmed by Western blot analysis and elevated immunoreactivity was identified through indirect ELISA. In conclusion, availability of the recombinant protein provides an effective system to study the immunological aspects of BoNT/F and corresponding applications in pathogen detection and vaccine candidacy. Keywords: Clostridium botulunium; Botulinum Neurotoxin Type F (BoNT/F) domains; cloning; recombinant protein expression; immunoreactivity DOI: http://dx.doi.org/10.3126/njb.v2i1.5634 Nepal Journal of Biotechnology Jan.2012, Vol.2(1): 1-15

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