Engineering Carboxylic Acid Reductase (CAR) via NADPH Recycling

Xuan Le Author

04/02/2021 Added

31 Plays

Description

Rapid evolution of enzyme activities is often hindered by the lack of efficient and affordable methods to identify beneficial mutants. This video discusses the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified E. coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries.

Searchable Transcript

Search:

[00:00:02.070]Hello, everyone.
[00:00:02.950]Welcome to my presentation.
[00:00:04.830]My name is Xuan Le, and I am an undergraduate student
[00:00:07.950]studying chemical engineering here at UNL.
[00:00:10.770]Today, I will be talking about the engineering
[00:00:13.180]of carboxylic acid reductase, or CAR enzymes,
[00:00:16.920]via NADPH recycling.
[00:00:21.010]A large number of chemical synthetic processes
[00:00:23.990]used in our world
[00:00:25.030]produce or depends on toxic materials.
[00:00:28.410]Enzymes are biological solutions
[00:00:30.280]to these traditional chemical synthesis
[00:00:32.670]because they catalyze useful reactions
[00:00:35.550]and usually require only mild reaction conditions.
[00:00:39.910]CAR enzymes selectively reduce carboxylic acids
[00:00:43.570]into their corresponding aldehydes.
[00:00:46.540]Aldehydes are mainly used as perfumes, flavoring agents,
[00:00:50.550]and they can also be easily converted
[00:00:53.220]into other products such as alcohols and amines,
[00:00:56.840]which can be used in polymer production.
[00:01:00.050]Figure 1 shows the domain architecture
[00:01:02.300]and catalytic cycle of CAR.
[00:01:04.700]CAR has three domains: an adenylation domain shown in blue,
[00:01:09.540]a reduction domain shown in red,
[00:01:12.030]and an internal phosphopantetheine attachment domain
[00:01:15.840]shown in orange.
[00:01:17.940]CAR uses ATP and NADPH as cofactors.
[00:01:23.360]The chemical reactants of enzymes are called substrates,
[00:01:26.810]and previous works have shown that CARs
[00:01:29.470]have broad substrate specificities.
[00:01:32.651]What this means is that CAR can be used
[00:01:34.740]to synthesize a wide range of chemicals.
[00:01:37.610]However, CARs have poor kinetic properties
[00:01:40.440]on substrates of interest, mainly dicarboxylic acids
[00:01:44.200]like adipates, that are common in nature.
[00:01:48.060]In this work, an NADPH-overaccumulating host strain,
[00:01:52.140]delta-2 was designed.
[00:01:54.280]This strain was used as a selection platform
[00:01:57.070]to identify CAR mutants with improved kinetic properties
[00:02:00.590]on the desired substrate adipate.
[00:02:03.570]Some experimental procedures performed
[00:02:05.810]include single-site saturation mutagenesis,
[00:02:09.430]growth-coupled selection of CAR mutant libraries,
[00:02:12.410]protein purification, and biochemical assays.
[00:02:16.840]We started the engineering process
[00:02:18.930]by first constructing a suitable host,
[00:02:21.820]as shown in Figure 2A.
[00:02:23.860]An E. coli strain that overaccumulates NADPH
[00:02:27.520]was designed by deleting two genes,
[00:02:30.590]resulting in a new strain called delta-2.
[00:02:34.320]When cultured in minimal media containing glucose
[00:02:37.550]as the carbon source, we saw that delta-2
[00:02:40.490]had extended lag phase compared to wild-type E. coli,
[00:02:44.890]and this can be seen in Figure 2B.
[00:02:48.130]This curve labeled "wt" represents the growth curve
[00:02:52.450]of the wild type.
[00:02:53.980]And this gray curve represents that of delta-2.
[00:02:58.460]And you can see clearly that delta-2
[00:03:00.690]had a much longer lag phase comparing to the wild type.
[00:03:05.900]We hypothesize that delta-2's long lag phase
[00:03:08.910]was because of toxic overaccumulation of NADPH.
[00:03:13.340]And in order to fix this,
[00:03:15.200]we introduced the Mycobacterium avium CAR, or MavCAR,
[00:03:20.040]into delta-2.
[00:03:21.820]As a reminder, CAR uses NADPH,
[00:03:25.270]so it will consume the overaccumulated NADPH
[00:03:29.510]when expressed in delta-2 in the presence
[00:03:32.500]of a carboxylic acid substrate.
[00:03:35.920]As shown in Figure 2B, this was the case.
[00:03:40.005]MavCAR-delta-2 had a much shorter lag phase
[00:03:44.560]comparing to delta-2 in the presence benzoate,
[00:03:48.830]which is a native substrate of CAR.
[00:03:52.150]This result confirms
[00:03:53.810]that the CAR-catalyzed reaction
[00:03:56.080]recovered the growth of delta-2 cells.
[00:03:59.980]Next, we looked at the correlation
[00:04:02.070]between growth rate of delta-2
[00:04:04.330]and the activity level of CAR.
[00:04:06.920]Cells were cultured on media plates
[00:04:09.250]containing glucose as the carbon source
[00:04:11.980]and different concentrations
[00:04:13.570]of 6-hydroxyhexanoate substrate.
[00:04:16.990]Looking at Figure 3,
[00:04:18.810]number 1 represents delta-2,
[00:04:21.060]in which there is no growth.
[00:04:23.570]Number 4 represent wild type,
[00:04:25.820]which acted as a positive control.
[00:04:28.930]And most importantly, number 2 and 3
[00:04:31.520]represent growth of delta-2 strains expressing MavCAR.
[00:04:36.460]At higher concentrations, we saw faster growth.
[00:04:40.940]This positive correlation
[00:04:42.500]between CAR activities and cell growth
[00:04:44.810]demonstrated that E. coli delta-2
[00:04:47.433]is an appropriate host for detecting CAR variants
[00:04:51.170]with improved activities.
[00:04:54.520]So to determine if cell growth could be used
[00:04:57.460]to identify MavCAR mutants with improved kinetic properties,
[00:05:02.100]we generated mutants at the MavCAR active site,
[00:05:05.230]which is the region responsible
[00:05:07.080]for binding the carboxylic acid substrate.
[00:05:10.250]The 10 residues labeled in black in Figure 4 were mutated
[00:05:14.200]because we presumed that they were important
[00:05:16.690]for binding of desired substrate adipate.
[00:05:20.350]We transformed the delta-2 host
[00:05:22.320]with each of the 10 libraries.
[00:05:24.870]Then we plated the transformed cells
[00:05:26.850]on media plates containing glucose and adipate.
[00:05:30.620]We monitored colony formation within a 72-hour period,
[00:05:35.010]and only colonies with faster growth rates
[00:05:37.870]than the control were picked.
[00:05:39.750]DNA sequencing analysis were then performed
[00:05:42.680]on these chosen colonies in order to identify mutations.
[00:05:47.300]The chosen mutants were expressed, purified,
[00:05:50.000]and characterized by kinetic assays.
[00:05:53.040]Table 1 shows kinetic activities
[00:05:55.330]of the chosen mutants on adipate.
[00:05:57.970]Catalytic efficiency kcat/ Km
[00:06:01.620]is the best value to represent the enzyme's overall ability
[00:06:05.510]to convert substrate to product.
[00:06:08.170]Most of the identified mutants had improved efficiency
[00:06:12.260]with the best mutant being N335R,
[00:06:16.370]as it had a 17-fold improvement in catalytic efficiency
[00:06:21.790]comparing to that of the wild type.
[00:06:24.840]The reason that this mutant performed so well
[00:06:27.520]could be due to electrostatic interaction.
[00:06:30.850]R is the amino acid arginine,
[00:06:33.130]and at a physiological pH of about 7.4,
[00:06:37.103]arginine's side chain is positively charged.
[00:06:40.590]At the same pH, the second carboxylic group of adipate
[00:06:44.620]is deprotonated and is negatively charged.
[00:06:48.200]So there is presumably an electrostatic interaction
[00:06:52.150]between these groups to improve substrate binding.
[00:06:56.260]The selection experiment, however,
[00:06:58.530]did lead to two false positives,
[00:07:01.740]T390G and G391R,
[00:07:05.920]which had no increased catalytic efficiency.
[00:07:10.650]In conclusion, these results show that the developed scheme
[00:07:14.550]is a successful strategy to identify CAR mutants
[00:07:17.390]with improved kinetic properties.
[00:07:19.650]In future work, this scheme could be applied
[00:07:22.030]to other substrates and other NADPH-oxidizing enzymes.
[00:07:26.910]And finally, I would like to acknowledge
[00:07:28.780]that this research would not have been possible
[00:07:31.170]without its UCARE award along with the grants
[00:07:34.150]from the National Science Foundation
[00:07:36.400]and the Nebraska Center for Energy Sciences Research.
[00:07:40.930]And I would like to thank my advisor, Dr. Niu,
[00:07:43.340]for being such a great mentor and resource.
[00:07:47.120]In addition, I would like to thank Levi Kramer,
[00:07:49.620]who was the grad student that has guided me
[00:07:52.260]throughout this project.
[00:07:53.800]He just graduated, so congratulations to him.
[00:07:57.700]And everyone in Niu's lab have been very helpful.
[00:08:00.930]I am very fortunate for being able to work with them.
[00:08:05.410]Thank you for listening and have a great rest of your day.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Engineering Carboxylic Acid Reductase (CAR) via NADPH Recycling

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments