ROBOX has received funding from the European Union (EU) project ROBOX (grant agreement n&#176; 635734) under EU&#39;s Horizon 2020 Programme Research and Innovation actions H2020-LEIT BIO-2014-1. The views and opinions expressed herein are only those of the author(s), and do not necessarily reflect those of the European Union Research Agency. The European Union is not liable for any use that may be made of the information contained herein.
The PhD thesis of J. Fischer is co-financed by a grant for &#34;Industrienahe Dissertation&#34; of the Austrian Funding agency FFG.
We thank Gernot John (PreSens GmbH) for the helpful discussions and valuable input for the manuscript draft.

1. Media Preparation
<ol>
	<li>Buffered minimal media (BMG with glycerol, BMD with dextrose as carbon source) preparation (1 L)
	<ol>
		<li>Autoclave 650 mL of distilled water in a 1 L screw cap glass bottle. After cooling down, complete the medium by adding 100 mL of autoclaved 10x YNB (134 g/L yeast nitrogen base w/o amino acids), 200 mL of potassium phosphate buffer (1 M, pH 6), 30-100 mL of 10% w/v glucose or glycerol (depending on the desired generated biomass amount during the batch), 2 mL of sterile 500x Biotin (10 mg/mL) solution and fill up to 1 L with sterile distilled water. 
		Note: All ingredients have to be autoclaved in separate flasks. Biotin is destroyed when autoclaved, and therefore has to be filter sterilized with 0.2 &#181;m membrane filters.</li>
	</ol>
	</li>
	<li>Buffered rich media (BYPG) (1 L)
	<ol>
		<li>Autoclave 700 mL of distilled water with 1% w/v yeast extract and 2% w/v peptone in a 1 L screw cap bottle. After cooling down, add 30-100 mL of the separately autoclaved 10% w/v glycerol, 200 mL of potassium phosphate buffer (1 M, pH 6) and fill up to 1 L with sterile distilled water.</li>
	</ol>
	</li>
</ol>
2. Shake Flasks Preparation
<ol>
	<li>Add 5 mL of distilled water in the 250 mL baffled shake flasks, cover it with two layers of cotton cloth and fix it in place with rubber bands. Cover the cloth with a piece of aluminum foil.</li>
	<li>Autoclave the flasks at 121 &#176;C for 20 min for full sterilization.</li>
	<li>Remove residual water from the flask and aliquot 50 mL of the previously prepared media to each flask.</li>
</ol>
3. Overnight Culture Inoculation
<ol>
	<li>Start an overnight culture (ONC) with a single fresh colony of the desired expression strain in 5 mL of YPD (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose) in a 50 mL centrifuge tube while leaving the lid slightly open to allow proper aeration.</li>
	<li>Let it grow over night at 28 &#176;C, 80% humidity, in a shaker at 100-130 rpm (2.5 cm shaking diameter).</li>
</ol>
4. Measurement Setup and Main Culture Cultivation
<ol>
	<li>For setting up the online monitoring system, follow the manufacturer&#8217;s instructions for appropriate calibration of the devices.</li>
	<li>Place a computer, where the appropriate software for monitoring is installed in range for a Bluetooth connection to the measurement stations. Start the software to connect the devices via Bluetooth.
	<ol>
		<li>Turn on the Bluetooth connection on the biomass measuring device by pressing the silver button on the front.</li>
		<li>In the software, click Devices then Find Devices. 
		Note: The devices should appear as a list in the window below the task line. If they do not, it is advisable to check if the batteries are charged and the computer is within reach for a Bluetooth connection.</li>
		<li>Drag and drop the found devices to the squares on the right side of the screen Measurement Tray.</li>
		<li>Click Connect. 
		Note: When the connection was successful, a control screen appears.</li>
		<li>To start a test run for the Bluetooth connection, click Measurement.</li>
		<li>Set up the experiment.
		<ol>
			<li>Click Start Measurement, name the experiment and enable all parameters needed to monitor. 
			Note: Licenses are needed for each of the possible measured parameters.</li>
			<li>Set Interval to 3 min. 
			Note: The interval determines how often measurement points are taken and every measurement point results in a value afterwards. Measuring every minute is very accurate, but lots of data are generated which makes it more laborious to evaluate.</li>
			<li>Set Average Measurement Points to 11. 
			Note: This setting determines how many measurement points are actually taken every 3 min. Therefore, the output is the mean value of 11 measurement points over 11 s.</li>
			<li>Enter the names for the samples.</li>
			<li>Skip the next screen, as the angle was already calibrated before (Step 4.1). 
			Note: A test run for the Bluetooth connection before inoculating the main cultures is advised to ensure stable connections and appropriate location of the connected computer. Also, the batteries have to be charged.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measure the cell density of the ONC (Step 3.1) diluted in cultivation media (1:20) in a spectrophotometer at 600 nm wavelength. Inoculate 50 mL of the medium (recommended carbon source concentration 0.3-1%, different media possible as described in Step 1.) with the ONC to an OD600 of 0.05 by using the following calculation. 
	<img alt="Equation 1" src="/files/ftp_upload/58589/58589eq1.jpg" /></li>
	<li>Place the flasks in the detectors and shake at 28 &#176;C, 130 rpm and 80% humidity. 
	Note: It is very important to give the culture 5 min of shaking before starting the measurement to avoid cells sticking to the bottom of the flask and yielding wrong values.</li>
	<li>Click Start Measurement in the software.</li>
	<li>Take 0.2 mL samples for cell density measurement 4 h after the inoculation and when the carbon source gets depleted (determination described in step 4.7) in triplicates. Dilute the samples appropriately in cultivation media (first measurement 1:5, second measurement 1:20) and measure the absorption at 600 nm wavelength in a spectrophotometer. Before removing the flasks and even before stopping the shaker, always pause the measurement in the software. 
	Note: The detectors are calibrated for recording the parameters under defined shaking conditions and will yield to nonsense measurements when recording still cultures. Also, make sure to always wait 5 min of shaking before starting the measurement again. The cell density measurements are essential because the detector is delivering just values for the biomass and not real cell density. A calibration function between the OD600 values (x values) and the values obtained by the online measurement system (y values) can be achieved by measuring several timepoints spectrophotometrically. The values are not in a linear but in a logarithmic relation according to the following calibration function: 
	<img alt="Equation 2" src="/files/ftp_upload/58589/58589eq2.jpg" /> 
	y: OD600 values obtained by spectrophotometer 
	x: values obtained by online measurement system 
	k: slope 
	d: axis intercept 
	The slope and the axis intercept can then be used to convert any value into the corresponding OD600 value. In Excel one can plot the spectrophotometrically obtained OD600 values against the values from the online system from the same timepoint. The addition of a logarithmic trendline and the corresponding equation will give the calibration function shown above.</li>
	<li>Cultivate until the carbon source is nearly depleted (app. 12-20 h). For that purpose, let the cultures grow until it can be seen in the software diagrams that the oxygen concentration is approaching zero and the cells are in the exponential growth phase. 
	Note: This is the case for carbon source concentrations used in this protocol. The timepoint of carbon source depletion can be determined with the online monitoring system as it is shown exemplarily in Figure 1 in the Representative Results section, when the oxygen level approaches zero and the cells are in the exponential growth phase.</li>
</ol>
5. Protein Expression by De-repression
<ol>
	<li>When the timepoint described in Step 4.6 is reached, start feeding the cultures by the addition of four feed discs per flask under sterile conditions. 
	Note: Under the described conditions, four glycerol feed discs release 2 mg/h glycerol according to the manufacturer.</li>
	<li>Continue the cultivation for 60-90 h while taking samples every 24 h to monitor the protein expression by an assay of choice (e.g., Bradford assay15, SDS PAGE16 or activity assays) and cell density measurements.</li>
</ol>
6. Culture Harvesting
<ol>
	<li>After the 60-90 h of cultivation, fill the cultures in 50 mL centrifuge tubes for harvesting by centrifugation at 4,000 x g, 4 &#176;C for 10 min. 
	Note: The supernatant (for secreted proteins) or the cells (for intracellular protein expression) can be harvested by centrifugation at 4,000 x g, 4 &#176;C for 10 min and further analyses can be done.</li>
	<li>Export the online measurement data as a spreadsheet file from the software for further analysis.</li>
	<li>Fill 5-10 mL of distilled water to all the flasks and autoclave to inactivate the remaining cells.</li>
</ol>

<ol>
	<li>Bill, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Playing+catch-up+with+escherichia+coli:+Using+yeast+to+increase+success+rates+in+recombinant+protein+production+experiments.">Playing catch-up with escherichia coli: Using yeast to increase success rates in recombinant protein production experiments.</a> Frontiers in Microbiology. 5 (MAR), 1-5 (2005).</li><li>Cregg, J. M., Cereghino, J. L., Shi, J., Higgins, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+Protein+Expression+in+Pichia+pastoris).">Recombinant Protein Expression in Pichia pastoris).</a> Molecular Biotechnology. 16, 23-52 (2000).</li><li>Weinhandl, K., Winkler, M., Glieder, A., Camattari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+source+dependent+promoters+in+yeasts.">Carbon source dependent promoters in yeasts.</a> Microbial Cell Factories. 13, 5 (2014).</li><li>Vogl, T., Kickenweiz, T., Sturmberger, L., Glieder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+Promoter.">Bidirectional Promoter.</a> US patent. , (2005).</li><li>Vogl, T., Glieder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Pichia+pastoris+promoters+and+its+consequences+for+protein+production.">Regulation of Pichia pastoris promoters and its consequences for protein production.</a> New Biotechnology. 30 (4), 385-404 (2013).</li><li>Ru&#223;mayer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems-level+organization+of+yeast+methylotrophic+lifestyle.">Systems-level organization of yeast methylotrophic lifestyle.</a> BMC Biology. 13 (1), 80 (2015).</li><li>Sakai, Y., Yurimoto, H., Matsuo, H., Kato, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+peroxisomal+proteins+and+organelle+proliferation+by+multiple+carbon+sources+in+the+methylotrophic+yeast,+Candida+boidinii.">Regulation of peroxisomal proteins and organelle proliferation by multiple carbon sources in the methylotrophic yeast, Candida boidinii.</a> Yeast. 14 (13), 1175-1187 (1998).</li><li>Hartner, F. S., Glieder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+methanol+utilisation+pathway+genes+in+yeasts.">Regulation of methanol utilisation pathway genes in yeasts.</a> Microbial Cell Factories. 5, 39 (2006).</li><li>Vogl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Toolbox+of+Diverse+Promoters+Related+to+Methanol+Utilization:+Functionally+Verified+Parts+for+Heterologous+Pathway+Expression+in+Pichia+pastoris.">A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris.</a> ACS Synthetic Biology. 5 (2), 172-186 (2016).</li><li>Mellitzer, A., Weis, R., Glieder, A., Flicker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+lignocellulolytic+enzymes+in+Pichia+pastoris.">Expression of lignocellulolytic enzymes in Pichia pastoris.</a> Microbial Cell Factories. 11 (1), 61 (2012).</li><li>Macauley-Patrick, S., Fazenda, M. L., McNeil, B., Harvey, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+protein+production+using+the+Pichia+pastoris+expression+system.">Heterologous protein production using the Pichia pastoris expression system.</a> Yeast. 22 (4), 249-270 (2005).</li><li>Weis, R., Luiten, R., Skranc, W., Schwab, H., Wubbolts, M., Glieder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+high-throughput+screening+with+Pichia+pastoris+by+limiting+yeast+cell+death+phenomena.">Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena.</a> FEMS Yeast Research. 5 (2), 179-189 (2004).</li><li>Ukkonen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Improvement of Recombinant Protein Production in Shaken Cultures. , (2014).</li><li>Jeude, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fed-batch+mode+in+shake+flasks+by+slow-release+technique.">Fed-batch mode in shake flasks by slow-release technique.</a> Biotechnology and Bioengineering. 95, 433-445 (2006).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Smith, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS+Polyacrylamide+Gel+Electrophoresis+of+Proteins.">SDS Polyacrylamide Gel Electrophoresis of Proteins.</a> Methods in molecular biology. 1, 41-55 (1984).</li><li>Hartner, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter+Library+Designed+for+Fine-Tuned+Gene+Expression+in+Pichia+Pastoris.">Promoter Library Designed for Fine-Tuned Gene Expression in Pichia Pastoris.</a> Nucleic Acids Research. 36 (12), e76 (2008).</li><li>Panula-Per&#228;l&#228;, J., Vasala, A., Karhunen, J., Ojamo, H., Neubauer, P., Mursula, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+slow+glucose+feed+cultivation+of+Pichia+pastoris+without+repression+of+AOX1+promoter:+towards+high+throughput+cultivations.">Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations.</a> Bioprocess and Biosystems Engineering. 37 (7), 1261-1269 (2014).</li></ol>

Although the presented protocol is generally applicable for cultivation and induction under derepressed conditions, bisy e.U. declares to have an interest in commercialization of the new derepressed promoters used in this study.

This method presents a reliable protocol for efficient methanol independent inducible expression by P. pastoris as an alternative to classical methanol induced protein expression driven by PAOX112. De-repressible promoters, like the PDC, the PDF or previously described mutated PAOX1 variants17, build the molecular basis for this new protein production concept. The shown representative results underline the usefulness and practicability of recombinant protein expression in P. pastoris by de-repressed promoters and small-scale screening with simple online monitoring. On the one hand, the harmful and toxic methanol that is usually used for the induction of the AOX1 promoter can be eliminated from the cultivation. On the other hand, the growth and the protein production phase are uncoupled9 in contrast to constitutive protein expression, e.g., with the commonly used PGAP. This is especially beneficial when the cells should express toxic or harmful proteins that display a burden for them.
The possibility to start the cultivations with different carbon source concentrations enables the decision of how much biomass one would like to gain before the protein production phase starts. When choosing a lower carbon source concentration in the batch, oxygen transfer limitations due to too high cell densities can be reduced while on the other hand higher cell densities can also result in higher protein titers. By monitoring the culture development in terms of biomass generation and oxygen concentrations, the optimal time-point for fed-batch initiation can be determined easily as it is shown in Figure 1.
For online monitoring of these cultivation parameters, an appropriate measurement system should be applied. The biomass measuring device (e.g., SFR Vario) is a simple and economic online measurement system for culture monitoring in shake flasks. The optoelectronic reader determines cell growth in the flask via scattered light detection and reads out the signal of the oxygen sensor integrated in the flask. The optics for biomass measurement consist of an LED that emits a light signal, which is scattered by particles (cells) in the culture broth and detected with a photodiode. The optical sensors emit fluorescence when excited with light of a certain wavelength. Depending on the amount of analyte molecules present, this fluorescence signal changes, which is detected by the reader optics and translated into concentration values. However, a cell density measurement calibration has to be established beforehand, since the measurement system is not measuring actual OD600 values. The oxygen uptake rate can be calculated from the slope of the oxygen curve with the reader software, and the temperature as well as rpm are recorded continuously together with the other parameters. To enable monitoring with this system, the shake flasks have to be previously equipped with the appropriate sensors for the desired parameters.
By the addition of four slow-release polymer discs providing a constant amount of glycerol to the culture broth, biomass accumulation is nearly stopped, and recombinant protein production is continued. The determination of the feed disc addition time point is not a critical step in this experiment, but it should be considered that a premature feed start before the exponential growth phase could be inhibiting the promoter activation as the expression starts upon carbon source depletion. Additionally, the total feeding time due to polymer capacity limitations may be reduced, whereas delaying the feed after the cells have reached the stationary phase might lead to starving and degradation of already produced protein. The amount of feed discs used in this protocol was determined experimentally for the optimal carbon source release to keep the promoter de-repressed while holding biomass generation low (data not shown). In this manner, the cultivations can be performed easily over 160-180 h without any intervention.
Main applications of this new protocol will be in expression clone screening, enzyme evolution and the discovery, the characterization and engineering of new promoters employing a methanol-free cultivation protocol in well-monitored conditions. In principle, the same protocol should be applicable for mixed-feed strategies employing methanol and limited fermentative carbon source feeds such as described by Panula-Per&#228;l&#228; et al. in 201418.

Improvement in yield and reliable cultivation conditions on large scale for recombinant protein production by microorganisms such as bacteria or yeast is one of the major needs of today&#39;s biotechnology1 and the commercial success of industrial applications. Dueto simple cultivation procedures and media, high yields and well characterized tools2 Pichia pastoris (Komagataella phaffii) is a widely used non-conventional yeast for recombinant protein production. Additional innovative tools are constantly developed for this species, like new expression vectors including new promoters as alternatives to the widely used PAOX1, the promoter region of the alcohol oxidase 1 gene3,4,5. A 500 bp long DNA sequence upstream of the CAT1 (CTA1) gene was identified as the promoter region, which enables a new methanol-free and versatile expression strategy in P. pastoris. The CAT1 gene is the only catalase gene of P. pastoris and the protein is located in the peroxisomes. The catalase plays an important role in the detoxification of reactive oxygen species, which are arising, e.g., from methanol oxidation in the peroxisomal MUT pathway or during beta oxidation of fatty acids6,7. The expression of the CAT1 gene is repressed in the prescence of glucose or glycerol in the cultivation medium and derepressed upon the depletion of these carbon sources8. Moreover, the expression of the catalase gene can be induced with methanol and remarkebly also with oleic acid, seemingly being the only gene of the P. pastoris MUT pathway that can be induced with oleic acid even reaching similar expression levels as with methanol induction9.
The 500 bp fragment of this P. pastoris CAT1 promoter, which is called PDC (sometimes also abbreviated PCAT1-500), has already been tested for the production of intracellularly expressed and secreted proteins and it proved to be an attractive alternative to the two classical P. pastoris promoters &#8211; the strong constitutive PGAP and the methanol inducible PAOX1, which were developed more than 20 years ago. The PDC was able to reach 21% (CalB) and 35% (HRP) of the volumetric activities compared to the PGAP in sugar rich medium. Upon methanol induction, the PDC did not only respond faster than the PAOX1, but could also clearly outperform it by a 2.8 fold higher final titer of CalB9.
Besides the PDC, an orthologous promoter (PDF) had been identified exhibiting a similar regulation profile. However, it is significantly stronger than the PDC under derepressed as well as under methanol-induced conditions (manuscript in preparation)3.
In cases where the strong constitutive PGAP failed because of physiologically problematic or cytotoxic proteins10,11, the PDC and the PDF represent an ideal alternative. In micro scale experiments, the expression driven by these promoters starts after the depletion of the initial carbon source (e.g., glucose or glycerol), which facilitates the biomass production without burdening the cells with the overexpression of the recombinant protein right from the beginning. While both of these promoters are still inducible by methanol, the activation by derepression makes additional use of the preinduction phase compared to the expressions employing PAOX1. Moreover, the PDC and PDF can be used for methanol-free protein production, which is a desirable goal for large industrial processes12.
For the development of applicable production strains and expression constructs, several steps in the development have to be passed in order to narrow down from large numbers of transformants to the preferred candidate for scale-up. Screenings usually are performed in high throughput in multi-well plates (micro scale: less than 0.5 mL) in order to capture a range of clones as large as possible. Subsequent re-screening and re-re-screening is the next step, followed by shake flask (small scale: 50-250 mL) or (micro-) bioreactor screenings12. However, it is desireable to have a method, which is most similar between the different scales from hundreds of microlitres in deep-well plates up to several milliliters in shake flasks up to later scale-up of methanol-free production in well-controlled bioreactors. Although shake flasks can provide already similar volumes as small bioreactors, there are several limitations, which are highly relevant for large scale protein production such as constant feeding, aeration and online-monitoring13 of growth and oxygen supply. Optical monitoring employing adapted shake flasks and shaking devices provide a cost-effective alternative to more sophisticated equipment for parallel cultivation while still providing constant online information about major culture parameters such as biomass and dissolved oxygen concentration. Slow release of carbon source from polymer carriers can be applied to secure a constant and controlled feed without relying on complex technical solutions and devices, simulating more similar conditions of bioreactors than the previously applied simple full depletion of carbon source9,10, where energy for ongoing protein expression becomes limiting.
The following method describes a small to medium scale methanol-free controllable protein expression system in P. pastoris based on the new promoters PDC and PDF. Induction by derepression involves two phases. A first short phase of biomass accumulation without the production of the protein of interest, using a carbon source that represses the promoters and a second phase of target protein production, where a carbon source is supplied in small but constant concentrations maintaining the promoter derepressed. Online monitoring of most critical cultivation parameters is applied during the whole cultivation period. The goal was to provide a methanol free, reliable and scalable cultivation system for P. pastoris.

<table><tbody><tr><td>Bacto Yeast Extract</td><td>BD Biosciences</td><td>212720</td><td></td></tr><tr><td>Baffled Flask 250 mL</td><td>DWK Life Science</td><td>212833655</td><td></td></tr><tr><td>BBL Phytone Peptone</td><td>BD Biosciences</td><td>298147</td><td></td></tr><tr><td>BioPhotometer</td><td>Eppendorf AG</td><td>5500-200-10</td><td></td></tr><tr><td>Certoclav-EL</td><td>CertoClav GmbH</td><td>9.842 014</td><td></td></tr><tr><td>Difco Yeast nitrogen base w/o amino acids</td><td>BD Biosciences</td><td>291920</td><td></td></tr><tr><td>Feed discs glycerol</td><td>Adolf Kuhner AG</td><td>315060</td><td></td></tr><tr><td>Glucose-monohydrate</td><td>Carl Roth GmbH + Co. KG</td><td>6887</td><td></td></tr><tr><td>Glycerol &amp;ge;98 %</td><td>Carl Roth GmbH + Co. KG</td><td>3783</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth GmbH + Co. KG</td><td>6875</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth GmbH + Co. KG</td><td>3904</td><td></td></tr><tr><td>Multitron Standard&amp;nbsp;</td><td>Infors AG</td><td>444-4226</td><td></td></tr><tr><td>SFR Vario v2</td><td>PreSens GmbH</td><td>200001689</td><td></td></tr></tbody></table>

methanol independent expression by pichia pastoris employing de-repression technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

As described in the protocol section, the software records the important cultivation parameters over the whole cultivation. Figure 1 shows the visualization of biomass development and oxygen concentration during a cultivation started with either 0.5 % (Figure 1A) or 0.3 % (Figure 1B) carbon source in the batch followed by the addition of glycerol feed discs. The online monitoring system is especially useful for the de-repressed cultivations as the timepoint of carbon source depletion after the batch can be determined exactly. As it can be seen in Figure 1, the oxygen concentration in the medium is decreasing rapidly and approaching zero, especially for 0.5 % glycerol in the batch (Figure 1A) while the biomass is increasing exponentially at this point. Here, the cells are in the exponential growth phase and in order to make optimal use of the carbon source release, shortly before the culture reaches the stationary phase, the feed discs should be added as it is described in the protocol. The addition of the feed discs before the oxygen concentration reaches zero is important for de-repressed protein expression to prevent the cells from oxygen limitation. Lower glycerol concentrations reduce the effect of short-term oxygen limitations as it can be seen in Figure 1B and are therefore recommended for de-repressed protein expression.
In the following, the results of two different cultivation experiments &#8211; employing the described protocol and the monitoring system shown in Figure 1&#160;&#8211; are described. In the first experiment, a P. pastoris strain producing the glucose oxidase from Aspergillus niger (A. niger) under the control of the PDC and in the second a P. pastoris strain producing the human growth hormone under control of the PDF is shown.
In this experiment, glucose oxidase production under the control of PDC was investigated. Therefore, different cultivation strategies were compared: glycerol pulsing, constant glycerol feed by addition of feed discs and cultivation without any feed or pulsing. The cultivation was done in 50 mL buffered minimal media with 0.5% glucose as sole carbon source in 250 mL shake flasks (Table 1).
Upon 160 h cultivation, the highest biomass concentrations were obtained, when a feeding strategy using a glucose batch for biomass production (38 h) and glycerol pulsing (0.25% w/v glycerol after 38 h, 61 h and 86 h cultivation) was employed (0.21 g/L total secretory protein, 8.4 U/mL). Although a 2-fold reduction of the biomass concentration and the total amount of secreted protein were obtained, when the feed disks were added after 38 h glucose batch, the volumetric activity was even 1 U/mL higher compared to the cultivation using glycerol pulsing. This indicates that a substantial amount of secreted protein does not contribute to the volumetric activity in the supernatant and may be secreted in an inactive form. Therefore, higher glycerol concentrations from glycerol pulses seemingly favor the formation of biomass instead of target protein production and constant low glycerol release led to more than two-fold higher specific productivity.
Another experiment employing the de-repressed expression method was done by expressing the human growth hormone (hGH) under control of PDF. The construct was stably integrated into the genome of P. pastoris strain BSYBG11. Here, 50 mL of BYPG, buffered rich medium, in 250 mL baffled shake flasks was used with 0.3% (w/v) glycerol as carbon source in the batch. Upon glycerol depletion, the protein expression with constant glycerol feed was ensured by the addition of 4 feed discs per flask and compared to no glycerol feed (Figure 2).
Figure 2 shows the comparison between hGH expression and biomass development of the same strain cultivated with and without constant glycerol feed. As assay sandwich ELISA was used with the secondary antibody fused to HRP and detection was carried out with 3,3&#8242;,5,5&#8242;-tetramethylbenzidine (TMB) as substrate. Especially for this protein, it seemed to be the case that without any feeding, the cells start to degrade the produced protein after approximately 30 h (light blue squares), whereas by feeding, this degradation could be prevented (dark blue triangles) and even the protein concentration per biomass still increased after 150 h of cultivation. As already observed for Hansenula polymorpha14, this result shows how switching from batch, like it is usually used in shake flask scale, to fed-batch mode can optimize P. pastoris cultivations.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58589/58589fig1.jpg" /> 
Figure 1: Visualization of online recorded parameters during two cultivations with different glycerol concentrations in the batch. The cultivations were started with 0.5% w/v (A) and 0.3% w/v (B) glycerol in the batch followed by the de-repression phase where the cells were fed by applying 4 feed discs per flask (release rate of 0.5 mg/h/disc according to the manufacturer). The dashed lines show the oxygen concentration in the medium, whereas the continuous lines show the cell density (OD600). Error bars show the standard deviation of six replicates (2 biological, three technical each). <a href="https://www.jove.com/files/ftp_upload/58589/58589fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58589/58589fig2.jpg" /> 
Figure 2: Comparison of human growth hormone (hGH) expression in batch and fed-batch mode. hGH content is indicated by absorption values at 405 nm by an ELISA where the 2nd antibody was an HRP fusion and TMB was used as a substrate for detection. Shown are the results for two different cultures: light blue line and squares display the cell density and hGH concentration normalized by the cell densities of a culture without any feed after the batch, compared to a culture, where the glycerol feed discs were applied (dark blue lines and triangles). Error bars show the standard deviation of six replicates (2 biological, three technical each). <a href="https://www.jove.com/files/ftp_upload/58589/58589fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">PDC-GOX at 160 h cultivation time</td>
			<td colspan="2">OD600</td>
			<td colspan="2">vol. activity [U/mL]</td>
			<td colspan="2">total amount of secreted protein [mg/mL]</td>
			<td>volumetric activity per cell density</td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>SD</td>
			<td>Mean</td>
			<td>SD</td>
			<td>Mean</td>
			<td>SD</td>
			<td>Mean</td>
		</tr>
		<tr>
			<td>BMD1% batch + glycerol pulsing</td>
			<td>74.6</td>
			<td>2</td>
			<td>8.4</td>
			<td>2.4</td>
			<td>0.21</td>
			<td>0.01</td>
			<td>0.11</td>
		</tr>
		<tr>
			<td>BMD1% batch + constant glycerol feed</td>
			<td>32.5</td>
			<td>0.3</td>
			<td>9.4</td>
			<td>0.7</td>
			<td>0.09</td>
			<td>0.01</td>
			<td>0.29</td>
		</tr>
		<tr>
			<td>BMD1% batch</td>
			<td>18.8</td>
			<td>0.6</td>
			<td>3.7</td>
			<td>0.7</td>
			<td>0.01</td>
			<td>0</td>
			<td>0.2</td>
		</tr>
	</tbody>
</table>
Table 1: Results of methanol independent GOX expression under the control of the de-repressed promoter PDC.

Watch this Scientific Journal Video about Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies at JoVE.com

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

methanol-independent-expression-pichia-pastoris-employing-de

Research

JoVE Journal

Bioengineering

13.5K Views.  bisy e.U.. This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Video: Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

Genetics

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Chemistry

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Biolistic Transformation of Yeast Mitochondria for Mutagenetic Analysis

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Author Spotlight: Advancing Techniques and Discoveries in Protein Synthesis and Assembly Through Innovative Mitochondrial Research

This work explains how to transform yeast mitochondria using a biolistic method. We also show how to select and purify the transformants and how to introduce the desired mutation in the target position within the mitochondrial genome.

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Biology

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Light-Controlled Fermentations for Microbial Chemical and Protein Production